Mpl ligand analogs

ABSTRACT

Mpl ligand analogs having one or more changed glycosylation sites as compared to a naturally occurring mpl ligand sequence of a corresponding number of amino acids are disclosed. The invention also relates to DNA sequences encoding mpl ligand analogs, recombinant plasmids and host cells for analog expression, and therapeutic compositions including such analogs.

This application is a continuation-in-part of U.S. Ser. No. 08/591,070,filed Feb. 9, 1996, now U.S. Pat. No. 5,756,083, which is acontinuation-in-part of U.S. Ser. No. 08/388,779, filed Feb. 15, 1995,now U.S. Pat. No. 5,696,250, which is a divisional application of U.S.Ser. No. 08/451,086, filed May 25, 1995, each of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to mpl ligand analogs having at least onechanged O- or N-linked glycosylation site. The invention also relates toDNA sequences encoding these mpl ligand analogs, and recombinantplasmids and host cells for analog expression.

BACKGROUND OF THE INVENTION

MGDF, or megakaryocyte growth and development factor, is a recentlycloned cytokine that appears to be the major regulator of circulatingplatelet levels. See Bartley, T. D. et al., Cell 77: 1117-1124 (1994);Lok, S. et al., Nature 369: 565-568 (1994); de Sauvage, F. J. et al.,Nature 369: 533-538 (1994); Miyazake, H. et al., Exp. Hematol. 22: 838(1994); and Kuter, D. J. et al., PNAS USA, 91: 11104-11108 (1994). MGDFas described in Bartley, T. D. et al., Cell 77: 1117-1124 (1994), isalso referred to as thrombopoietin (TPO), mpl ligand, and megapoietin.Herein, the term "mpl ligand" will be used generically to refer to allpolypeptides that activate the mpl receptor, including TPO and MGDF. Thempl receptor is a cell surface protein that, upon activation, leads toproduction and/or development of megakaryocytes and platelets. See WO92/07074.

"Mpl ligand analogs" are polypeptides that differ from native sequencesin a way that affects the number, location or type of carbohydratelinkage sites. Such polypeptides are one aspect of the presentinvention. Mature native human mpl ligand is a protein having 332 aminoacids in total. The sequence of this protein (attached to a 21-aminoacid long leader sequence) and the corresponding cDNA are shown in FIGS.1A-1C herein (SEQ. ID NOs.: 1 and 2).

Recombinant mpl ligand produced in both Chinese Hamster Ovary (CHO) andE. coli cells has been demonstrated to have a biological activity ofspecifically stimulating or increasing megakaryocytes and/or plateletsin vivo in mice, rats and monkeys. See e.g., Hunt, P. et al., Blood84(10): 390A (1994). Human mpl ligand molecules that have been truncated(as compared to the 332 amino acid protein encoded by the cDNA inhumans) so that they extend at least 151 amino acids, starting fromamino acid position 22 in FIG. 1A, retain biological activity in vivo.FIGS. 2A-2B (SEQ. ID NOs.: 3 and 4) shows one example of a truncated mplligand molecule which, in mature form, has 174 amino acids and hasbiological activity. In FIG. 2A, the 174 amino acid long protein isshown attached to a 21 amino acid N-terminal leader sequence. Thismolecule was used to create some of the mpl ligand analogs in theexamples section below. Other analogs are based on amino acids 1-199,1-191, and 1-183 of FIG. 1A-1C. It is also possible to remove up to thefirst six amino acids at the N-terminus of the mature human sequence mplligand protein and retain biological activity. Therefore, it appearsthat biological activity is retained within amino acids 7 to 151(inclusive) of the mature amino acid sequence of human mpl ligand shownin FIG. 1A-1C.

In general, many cell surface and secretory proteins produced byeukaryotic cells are modified with one or more oligosaccharide groups.This modification, referred to as glycosylation, can dramatically affectthe physical properties of proteins and can also be important in proteinstability, secretion, and subcellular localization. Proper glycosylationcan be essential for biological activity. In fact, some genes fromeukaryotic organisms, when expressed in bacteria (e.g., E. coli) whichlack cellular processes for glycosylating proteins, yield proteins thatare recovered with little or no activity by virtue of their lack ofglycosylation.

Glycosylation occurs at specific locations or sites along thepolypeptide backbone and is usually of two types: O-linkedoligosaccharides are attached to serine (Ser) or threonine (Thr)residues while N-linked oligosaccharides (chains) are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where x can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids not countingproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (hereafterreferred to as sialic acid). Sialic acid is usually the terminal residueof both N-linked and O-linked oligosaccharides and, by virtue of itsnegative charge, may confer acidic properties to the glycoprotein.

As used herein glycosylation "sites" are amino acid residues that arestructurally able to link to glycosyl residues, although such sites mayor may not be actually linked to a glycosyl residue. As noted above,O-linked sites are either Ser or Thr residues, whereas N-linked sitesare either Asn-X-Ser or Asn-X-Thr, where X is defined as any amino acidother than Pro (preferably one of the 19 naturally-occurring aminoacids, excluding Pro). Whether a given site is glycosylated with aglycosyl chain is determined by the host cell in which the molecule isexpressed, the amino acids neighboring the site, and other factors.

As used herein, the number of "chains" attached to a given mpl ligandanalog will be the average number of carbohydrate (i.e., glycosyl)chains attached to a given mpl ligand molecule expressed by a particularhost cell. Notably, the glycosylation sites for natural andcorresponding recombinant mpl ligand will generally be the same, whereasthe number of chains will possibly vary depending upon whether theparticular host cell used for recombinant expression attaches glycosylchains to the same sites or not, as compared to the natural source.Herein, whenever a comparison is made between recombinant and naturalmpl ligand analogs, the same number of amino acids will be compared,regardless of whether the natural source actually produces an mpl ligandmolecule having that length. Thus, "natural" refers to the sequenceemployed in a particular species (such as human) rather than the lengthof the molecule actually expressed in such natural source.

Naturally occurring mpl ligand is a glycosylated molecule. Theglycosylation pattern of natural mpl ligand is related to two keydomains that have been found in mpl ligand. The sequence of the firstapproximately 151 amino acids of mature human mpl ligand, correspondingto an active portion of the molecule, bears notable homology toerythropoietin (EPO), a cytokine capable of stimulating production oferythrocytes, and is referred to as the "EPO-like" domain of human mplligand. The remaining amino acids of the mature protein make up aso-called "N-linked carbohydrate" domain, since they include most if notall of the natural sites for N-linked glycosylation. In human mplligand, there are six N-linked glycosylation sites all contained in theN-linked glycosylation domain. Both domains contain O-linkedglycosylation sites. There are an estimated 12-14 O-linked glycosylationchains in the molecule. Experimental evidence with human mpl ligand DNAexpressed recombinantly in CHO cells reveals that in the EPO-like domainat least two O-linked sites are glycosylated, at positions 1 (Ser) and37 (Thr).

Glycoproteins such as mpl ligand can be separated into different chargedforms using techniques such as isoelectric focusing (IEF). For example,several parties have reported IEF studies of crude and partiallypurified erythropoietin preparations (Lukowsky et al., J. Biochem. 50:909 (1972); Shelton et al., Biochem. Med. 12: 45 (1975); Fuhr et al.,Biochem. Biophys. Res. Comm. 98: 930 (1981)).

In spite of the above information on glycosylation of mpl ligandmolecules, there remains a need to obtain mpl ligand molecules having adifferent glycosylation pattern and which retain or have improvedbiological activity.

Accordingly, it is an object of the present invention to provide novelglycosylated mpl ligand molecules, referred to as mpl ligand analogs. Itis a further object of this invention to provide pharmaceuticalcompositions containing such molecules and methods of treatingconditions treatable by mpl ligand with the mpl ligand analogs of thisinvention.

SUMMARY OF THE INVENTION

In one embodiment, the subject invention relates to analogs of mplligand comprising an amino acid sequence which includes at least oneadded, at least one deleted, and/or a combination of at least one addedand deleted, site for glycosylation as compared to the correspondingnatural sequence mpl ligand. The added or deleted site(s) forglycosylation may result in a greater or lesser number of carbohydratechains, and higher or lower sialic acid content, than correspondingnatural sequence mpl ligand, particularly human mpl ligand. For example,one type of analog could involve deleting one or more N- or O-linkedsites, and addition of one or more N- or O-linked sites at the same oranother position.

In another aspect of the above embodiment, the subject invention relatesto mpl ligand analogs comprising amino acid sequences which involvereplacement of one or more N- or O-linked glycosylation sites with oneor more non-naturally occurring sites. Thus, an N-linked site may bereplaced with a different N-linked site; an N-linked site may bereplaced with an O-linked site; an O-linked site may be replaced with adifferent O-linked site; and an O-linked site may be replaced with anN-linked site.

Combinations of any of the above changes are further encompassed withinthis invention.

The invention further encompasses DNA sequences encoding such mpl ligandanalogs, and recombinant plasmids and host cells for analog expression.

In all of the above cases, the change in glycosylation site results in achange in the number, amount, location or type (N- vs. O-) of glycosylchains in the resulting mpl ligand analog and retains a biologicalactivity of mpl ligand, i.e., the analog can still activate the mplreceptor. Activation of the mpl receptor means that megakaryocytopoiesisis enhanced thereby resulting in an increase in platelets in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows the DNA and amino acid sequence of native human mplligand including a signal peptide (amino acids -21 to -1) and the matureamino acid sequence (1-332).

FIG. 2A-2B shows the DNA and amino acid sequence of mpl ligandcorresponding to amino acids 1-174 of the human mature mpl ligandsequence attached to a 21 amino acid long signal peptide. The sequencesflanking the coding regions have introduced XbaI and SalI cloning sitesat the 5' and 3' ends respectively.

FIG. 3 shows a Western blot with E. coli and CHO expressed mpl ligand.MK stands for Met-Lys, which is added to the N-terminus of mpl ligandfor expression in E. coli, and may be cleaved off using a dipeptidase,such as cathepsin C. A molecule in which MK has been removed is referredto as desMK. Treatment with the glycosidases neuraminidase andO-glycanase is indicated.

FIG. 4 shows in vivo activity of E. coli and CHO expressed mpl ligand innormal mice, in terms of platelet counts. The data indicates thatglycosylated mpl ligand (CHO material) has superior activity thannon-glycosylated (E. coli) material. This may be a result of increasedhalf-life for the glycosylated material. For example, CHO 332 stands forhuman mpl ligand amino acids 1-332 (FIGS. 1A-1C) expressed in CHO cells.

FIG. 5 shows a Western blot analysis of COS cell supernatants ofrecombinant human mpl ligand and analogs 4, 6, 7, 9, 10, and 11. Theconstruction of the analogs is described in Example 4. Analogs 4, 7, 10have at least one additional carbohydrate chain as evidenced by slowergel mobility. The analog numbers correspond to analog numbers providedin Table 1 (e.g., 11 corresponds to analog N11). The control is N1 inTable 1.

FIG. 6 shows a Western blot analysis of COS cell supernatants ofrecombinant human mpl ligand and analogs 4, 5, 13, 14, and 15. Theconstruction of the analogs is described in Example 4. Analogs 4, 13,14, and 15 have at least one additional carbohydrate chain as evidencedby slower gel mobility.

FIG. 7 shows a Western blot analysis of COS cell supernatants of humanmpl ligand and indicated mpl ligand analogs after treatment withN-glycanase. The results indicate that the analogs have differentialglycosylation patterns.

FIGS. 8A-8D shows the results of a human megakaryocyte growth bioassaywith mpl ligand analogs. Panels A and D are the positive and negativecontrols respectively. The well pictured in panel A received 37.5 pg ofwild type (i.e., natural sequence) mpl ligand 1-174 COS-1 conditionedmedium and shows substantial megakaryocyte growth. Panel D received 1.5ul of COS-1 mock conditioned medium and shows no growth. Panels B and Care mpl ligand 1-174 analogs 7 and 10 respectively. Panel B received 9.0pg of mpl ligand COS-1 conditioned medium while panel C received 27 pgand both show excellent megakaryocyte growth.

FIG. 9 shows a Western blot analysis of CHO mpl ligand 1-174 and analogsN4 and N15 (see Table 1). Slower gel mobility demonstrates that analogN4 (4B) has one additional oligosaccharide while analog N15 (15-8) hastwo additional oligosaccharides.

FIG. 10 shows a Western blot of CHO cell-produced mpl ligand analogswith and without treatment with N-glycanase as indicated. Slower gelmobility after treatment with N-glycanase demonstrates the presence ofN-linked oligosaccharide.

FIG. 11 shows platelet counts from mice treated with various forms ofmpl ligand at various doses. The data demonstrate that increased amountsof N- and/or O-linked carbohydrate result in increased in vivo activity.

FIG. 12 shows a Western blot analysis of COS-produced mpl ligand 1-174,along with analogs N10, N15, N33, N39, N31, N35, and N40. The number ofadded N-linked glycosyl sites is also indicated. The figure shows thatincreasing the number of N-linked sites reduces the mobility of mplligand due to increasing amounts of N-linked carbohydrate.

FIG. 13 shows a Western blot analysis of COS-produced mpl ligand 1-174,along with analogs N15, N29, N30, and N38. The number of N-linkedglycosyl chains is also indicated.

FIG. 14 shows a Western blot analysis of CHO mpl ligands 1-174 and 1-199(N31) and analogs N40, and N43 (see Table 8). Gel mobility demonstratesthat analogs N40 and N43 have equal numbers of additionaloligosaccharide.

FIG. 15 shows a Western blot analysis of CHO mpl ligand 1-174 andanalogs N38, N41, and N42. Gel mobility demonstrates that each analoghas one additional oligosaccharide.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides mpl ligands with different glycosylationsites as compared to natural mpl ligand having a corresponding sequence.Preferably, the resulting molecules are those having additionalglycosylation sites that are occupied by glycosyl chains upon expressionin a mammalian cell (such as COS, CHO, and human cells).

In a first embodiment, the subject invention relates to analogs of mplligand comprising an amino acid sequence which includes at least oneadded, at least one deleted, and/or at least one added and deleted, sitefor glycosylation as compared to corresponding natural sequence mplligand. The added or deleted site(s) for glycosylation may result in agreater or lesser number of carbohydrate chains, and higher or lowersialic acid content, than corresponding natural sequence mpl ligand,particularly human mpl ligand. A combination of a deletion of one siteand addition of another site would result in no net change in the numberof sites, but rather, a change in location and/or type of site. Suchcombined change analogs are also encompassed within this invention.

In another aspect of the above embodiment, the subject invention relatesto mpl ligand analogs comprising amino acid sequences which includereplacement of one or more N- or O-linked glycosylation sites with oneor more non-naturally occurring sites. Thus, an N-linked site may bereplaced with a different N-linked site; an N-linked site may bereplaced with an O-linked site; an O-linked site may be replaced with adifferent O-linked site; and/or an O-linked site may be replaced with anN-linked site. Replacement of one site with another site in essentiallythe same location may have the result of increasing the glycosylationefficiency at that site, or other effects. For example, evidence isprovided herein that a Thr residue instead of a Ser residue may increasethe glycosylation efficiency at O-linked sites.

The term "mpl ligand", as used herein, includes naturally occurring mplligand, truncations of naturally occurring mpl ligand as well asnon-naturally occurring polypeptides having an amino acid sequence andglycosylation sufficiently duplicative of that of naturally occurringmpl ligand to allow possession of a biological activity of specificallystimulating growth, development and/or production of megakaryocytesand/or platelets. Mpl ligand analogs based on at least amino acids 7-151up to amino acids 1-332 of FIGS. 1A-1C are preferred.

In a preferred embodiment, mpl ligand is the product of the expressionof an exogenous DNA sequence that has been transfected into a eukaryotichost cell; that is, in a preferred embodiment the mpl ligand is"recombinant mpl ligand". The preferred eukaryotic host is mammalian,particularly preferably CHO cells. Recombinant mpl ligand isadvantageously produced according to the procedures described herein andin the publications cited herein regarding cloning and expression of mplligand.

Some additional preferred mpl ligand molecules have the following aminoacid sequences, based on FIGS. 1A-1C herein:

    ______________________________________                                        mpl ligand 1-332                                                                           amino acids 1-332 of FIGS. 1A-1C                                 mpl ligand 1-199                                                                           amino acids 1-199 of FIGS. 1A-1C                                 mpl ligand 1-191                                                                           amino acids 1-191 of FIGS. 1A-1C                                 mpl ligand 1-183                                                                           amino acids 1-183 of FIGS. 1A-1C                                 mpl ligand 1-174                                                                           amino acids 1-174 of FIGS. 1A-1C                                 mpl ligand 1-163                                                                           amino acids 1-163 of FIGS. 1A-1C                                 mpl ligand 1-153                                                                           amino acids 1-153 of FIGS. 1A-1C                                 mpl ligand 1-152                                                                           amino acids 1-152 of FIGS. 1A-1C                                 mpl ligand 1-151                                                                           amino acids 1-151 of FIGS. 1A-1C                                 mpl ligand 7-332                                                                           amino acids 7-332 of FIGS. 1A-1C                                 mpl ligand 7-199                                                                           amino acids 7-199 of FIGS. 1A-1C                                 mpl ligand 7-191                                                                           amino acids 7-191 of FIGS. 1A-1C                                 mpl ligand 7-183                                                                           amino acids 7-183 of FIGS. 1A-1C                                 mpl ligand 7-174                                                                           amino acids 7-174 of FIGS. 1A-1C                                 mpl ligand 7-163                                                                           amino acids 7-163 of FIGS. 1A-1C                                 mpl ligand 7-153                                                                           amino acids 7-153 of FIGS. 1A-1C                                 mpl ligand 7-152                                                                           amino acids 7-152 of FIGS. 1A-1C                                 mpl ligand 7-151                                                                           amino acids 7-151 of FIGS. 1A-1C                                 ______________________________________                                    

It should be noted, for example, that mpl ligand 1-183, 1-191, 7-183,and 7-191 encompass one or two additional naturally-occurringglycosylation sites on the C-terminus thereof, as compared to shortersequences. In each of the above cases, Met-Lys may further be includedin the N-terminus thereof.

The in vitro specific activities referred to herein are measurements ofrelative in vitro specific activities and are not measurements ofabsolute in vitro specific activities. For the purposes of thisapplication, the specific activities are used only to compare relativeactivities of mpl ligand analogs that have been assayed using the sameassay, using the same conditions including the same internal standard,and having the same analysis of the data used to calculate specificactivity, etc.

As used herein the phrases "analog of mpl ligand" or "mpl ligand analog"refer to mpl ligand with one or more changes in the amino acid sequenceof mpl ligand which result in a change in the type (N- or O-linked,which may affect the amount of carbohydrate attached), number, orlocation of sites for carbohydrate attachment. In a preferredembodiment, the change in glycosylation site(s) results in a change inthe number of glycosyl chains attached to the mpl ligand molecule. In aparticularly preferred embodiment, the change in glycosylation site(s)adds at least one (generally 1-6, preferably 1-5, particularlypreferably 2-4) glycosyl chains, and most preferably the chain(s)is(are) added via N-linkage. In another particularly preferredembodiment, the mpl ligand analog retains at least equivalent biologicalactivity in vivo as compared to natural sequence mpl ligand (e.g., humanmpl ligand) and may possess substantially higher activity in vivo, asmeasured in assays for biological activity. Such assays include thosethat detect megakaryocyte or platelet production.

To prepare such analogs of mpl ligand, preferably they are generated bysite-directed mutagenesis resulting in additions, deletions, orsubstitutions of amino acid residues that add, eliminate or alter sitesthat are available for glycosylation. By "altered" is meant that a sitehas been deleted while another has been added at the same or anotherlocation as the deleted site. However, as is appreciated by thoseskilled in the art, other methods could result in a gene encoding thesame amino acid sequence, and such methods are encompassed herein. Theresulting analogs may have fewer or more (preferably more) attachedcarbohydrate chains than natural human/recombinant mpl ligand.

Addition of one or more carbohydrate (i.e., glycosyl) chains to mplligand is one important object of this invention. Mpl ligand analogshaving more carbohydrate chains than those found in the correspondingnaturally-occurring amino acid sequence (e.g., 1-332 or 1-174, etc.) aregenerated by adding glycosylation sites which do not perturb thesecondary or tertiary conformation in a way that would substantiallyreduce biological activity. As used herein the "naturally-occurring" mplligand refers to an amino acid sequence having the corresponding numberof amino acids as the relevant analog, even if the particular length ofmpl ligand species is not actually expressed in the native species.Advantageously, the analog of mpl ligand has up to 6 additional sitesfor N-glycosylation or O-glycosylation, resulting in the addition offrom 1 up to 6 additional N-linked or O-linked carbohydrate chains (or acombination thereof).

For example, a Pro at position 30 is replaced by an Asn and a Val atposition 32 is replaced by a Thr to give the sequence Asn-Glu-Thr, whichserves as a new site for N-glycosylation (analog N4 below; see Table 1).

Analogs may also be constructed which have two or more additionalN-linked chains by combining mutations; for example, analogs N4 and N10described in Table 1 may be combined to yield an analog with twoadditional sites for carbohydrate addition (i.e., analog N15 in Table1). In a like manner analogs with three or more added chains can beconstructed. As will be appreciated by those skilled in the art, thesubject invention includes many other analogs of mpl ligand havingdifferent sites for glycosylation (in terms of number, type or locationof site). The mpl ligand analogs of this invention are in all casesparticularly preferably based on mpl ligand having a human amino acidsequence (see FIGS. 1A-1C and 2A-2B); however, analogs based on mplligand sequences from other species (e.g., dog, pig, monkey, mouse orrat) are also contemplated herein.

Insertions of amino acids to create glycosylation sites are alsocontemplated. For example, a Glu at position 57 is replaced by a Thr andAsn is inserted immediately after Met at position 55 as follows:##STR1## This adds a new glycosylation site (amino acids 55', 56, and57). See analog N23 below.

Also included within the analogs of this invention are analogs whichhave one or more amino acids extending from the carboxy terminal end ofmpl ligand wherein the carboxy terminal extension provides at least oneadditional carbohydrate site. The carboxy terminus of mpl ligand willvary depending upon the particular form of mpl ligand used (e.g., mplligand 1-332 amino acids, or mpl ligand 1-163 amino acids). Anadditional carbohydrate site may be added to the carboxy terminus of anmpl ligand species by adding amino acids to the carboxy terminus, suchamino acids containing one or more N- or O-linked glycosylation sites.

Tables 1 and 6 list some exemplary mpl ligand analogs which haveadditional sites for N-linked carbohydrate chains. The analogs have thesequence Asn-X-Ser or Asn-X-Thr included at various positions in thehuman mpl ligand polypeptide chain based on the human amino acidsequences to create N-linked sites. Tables 4 and 7 list those analogswhich add at least one additional N-linked carbohydrate chain, asevidenced by the migration of the glycoproteins on SDS gels (see,Example 6 and FIGS. 3, 5, 6, 7, 9, 10, 12, and 13). Note that theseTables also include some truncated species that are not "analogs" asdefined herein (i.e., N1, N16, N17, and N31). These are listed in theTables to show how various truncated species were prepared.

Also encompassed by the present invention are DNA sequences encoding thempl ligand analogs disclosed herein, preferably those encoding analogshaving additional sites for N-linked chains. Procedures used tointroduce changes into the mpl ligand DNA sequence for the purpose ofcreating, deleting and/or altering attachment sites for carbohydratesare disclosed in Examples 4 and 14.

These mpl ligand analogs can be the product of expression of anexogenous DNA sequence, i.e., produced through recombinant DNAtechnology, they can be chemically synthesized products or they may beproduced by combined methods. An exogenous DNA sequence comprises cDNA,genomic DNA or chemically synthesized DNA encoding an mpl ligand analog.Recombinant DNA plasmids and eukaryotic host cells useful for theexpression of such analogs are also provided. Expression vectors includeany vector which is capable of expressing cloned DNA sequences in aeukaryotic host cell, particularly those vectors used for expression inCOS and CHO cells. Examples of such vectors include plasmids pDSRα andpDSRα2, see Mol. Cell. Biol. 8: 466-472 (1988); WO 91/13160 (1991); andWO 90/14363 (1990). The cultivation of COS and CHO host cells expressingmpl ligand analogs was carried out using standard procedures known tothose skilled in the art.

Changing the number, type, location, or amount of carbohydrate chainsattached to mpl ligand may confer advantageous properties such asincreased solubility, greater resistance to proteolysis, reducedimmunogenicity, increased serum half-life, and increased or alteredbiological activity.

Conditioned media from COS cells expressing mpl ligand analogs N2-N15(N1 is human mpl ligand 1-174; see FIGS. 2A-2B) were analyzed for invitro biological activity and the results shown in Table 4.

Conditioned media from COS cells expressing mpl ligandanalogs/truncations N15-N40 were analyzed for in vitro biologicalactivity and the results are shown in Table 7.

In vivo biological activity results for various forms are presented inFIG. 11 (see Example 13).

Another embodiment of the invention relates to mammalian (e.g., ChineseHamster Ovary, CHO) host cells which preferentially synthesize mplligand or analogs of mpl ligand having greater than a specific number ofsialic acids per molecule, e.g. greater than that found in mpl ligand1-332, 1-199, 1-191, 1-183, 1-174, 1-163, 1-153, 1-152, or 1-151produced naturally or recombinantly in a eukaryotic cell. In vitroactivities of analogs N4 and N15, along with full-length and varioustruncated species expressed in CHO cells are shown in Table 5.

The sialic acid content of the mpl ligand molecule may affect its invivo biological activity. For example, tetraantennary (four-branched)N-linked oligosaccharides most commonly provide four possible sites forsialic acid attachment while bi- and triantennary oligosaccharides,which can substitute for the tetraantennary form at asparagine-linkedsites, commonly have at most only two or three sialic acids attached.O-linked oligosaccharides commonly provide two sites for sialic acidattachment. Thus, mpl ligand molecules with N-linked carbohydratesubstituted for O-linked carbohydrate can accommodate two additionalsialic acids per chain provided the N-linked oligosaccharides aretetraantennary. Mammalian cell cultures are screened for those cellsthat preferentially add tetraantennary chains to recombinant mpl ligand,thereby maximizing the number of sites for sialic acid attachment.

Dihydrofolate reductase (DHFR) deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins including recombinant mpl ligand.

Compositions comprising a therapeutically effective amount of an mplligand analog in accordance with this together with a suitable diluent,adjuvant and/or carrier useful in mpl ligand therapy are furtherencompassed by this invention. A "therapeutically effective amount" asused herein refers to that amount which provides therapeutic effect fora given condition and administration regimen.

The present compositions can be systemically administered parenterally.Alternatively, the compositions may be administered intravenously orsubcutaneously. When systemically administered, the therapeuticcompositions for use in this invention may be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such pharmaceutically acceptable protein solutions, with due regardto pH, isotonicity, stability and the like, is within the skill of theart. The specific route chosen will depend upon the condition beingtreated. The administration of mpl ligand or mpl ligand analogs ispreferably done as part of a formulation containing a suitable carrier,such as human serum albumin, a suitable diluent, such as a bufferedsaline solution, and/or a suitable adjuvant. The required dosage will bein amounts sufficient to raise the platelet levels of patients and willvary depending upon the severity of the condition being treated, themethod of administration used and the like.

The conditions to be treated by the methods and compositions of thepresent invention are generally those which involve an existingmegakaryocyte/platelet deficiency or an expected megakaryocyte/plateletdeficiency in the future (e.g., because of planned surgery). Thesecompositions and methods may also be useful to increase the number ofcirculating platelets in individuals to increase the number of plateletsavailable for donation. Such conditions will usually be the result of adeficiency (temporary or permanent) of active mpl ligand in vivo. Thegeneric term for platelet deficiency is thrombocytopenia, and hence themethods and compositions of the present invention are generallyavailable for treating thrombocytopenia.

Thrombocytopenia (platelet deficiencies) may be present for variousreasons, including chemotherapy, bone marrow transplants, and othertherapy with a variety of drugs, radiation therapy, surgery, accidentalblood loss, and other specific disease conditions. Exemplary specificdisease conditions that involve thrombocytopenia and may be treated inaccordance with this invention are: aplastic anemia, idiopathicthrombocytopenia, metastatic tumors which result in thrombocytopenia,systemic lupus erythematosus, splenomegaly, Fanconi's syndrome, vitaminB12 deficiency, folic acid deficiency, May-Hegglin anomaly,Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also,certain treatments for AIDS result in thrombocytopenia (e.g., AZT).Certain wound healing disorders might also benefit from an increase inplatelet numbers.

With regard to anticipated platelet deficiencies, e.g., due to futuresurgery or future thrombocytopenia-inducing therapy, an mpl ligandanalog of the present invention could be administered several days toseveral hours prior to the need for platelets. With regard to acutesituations, e.g., accidental and massive blood loss, an mpl ligandanalog could be administered along with blood or purified platelets.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the daily regimen should be in the range of 0.01-1000 micrograms of mplligand analog per kilogram of body weight, preferably 0.1-10 microgramsper kilogram of body weight.

The therapeutic methods, compositions and polypeptides of the presentinvention may also be employed, alone or in combination with othercytokines, soluble mpl (i.e., mpl ligand) receptor, hematopoieticfactors, interleukins, growth factors or antibodies in the treatment ofdisease states characterized by other symptoms as well as plateletdeficiencies. It is anticipated that an mpl ligand analog molecule willprove useful in treating some forms of thrombocytopenia in combinationwith general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Othermegakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor(SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or othermolecules with megakaryocyte stimulating activity may also be employedwith mpl ligand. Additional exemplary cytokines or hematopoietic factorsfor such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3,IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), GM-CSF,granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha(IFN-alpha), IFN-beta, or IFN-gamma. It may further be useful toadminister, either simultaneously or sequentially, an effective amountof a soluble mammalian Mpl receptor, which appears to have an effect ofcausing megakaryocytes to fragment into platelets once themegakaryocytes have reached mature form. Thus, administration of mplligand analog (to enhance the number of mature megakaryocytes) followedby administration of the soluble mpl receptor (to inactivate the analogand allow the mature megakaryocytes to produce platelets) is expected tobe a particularly effective means of stimulating platelet production.The dosage recited above would be adjusted to compensate for suchadditional components in the therapeutic composition. Progress of thetreated patient can be monitored by conventional methods.

Additional modifications of the analogs of this invention may also becarried out, e.g., to increase activity, stability, half-life, etc. Forexample, pegylation (poly- or mono-) could be added to the mpl ligandanalog via amino groups on the protein or via the carbohydrate groups.Also, fatty acids or other polymers could be attached to the protein orcarbohydrate groups.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.The mpl ligand standard used in the bioassays employed in the Examplesis a recombinant mpl ligand standard that was expressed in E. coli,refolded into an active conformation and purified. Thus, only relativespecific activities are being measured.

EXAMPLE 1 Construction of Mpl Ligand 1-174

Human mpl ligand gene encoding amino acids 1-174 (starting withS-P-A-P-P-A . . . ) of FIGS. 2A-2B was generated from a human fetalliver cDNA library (Bartley et al, Cell 77: 1117-1124 (1994) bypolymerase chain reaction (PCR). The 5' PCR primer encoded the aminoterminus of human mpl ligand, an XbaI site, and an optimized Kozaksequence. The 3' primer contained a termination codon and a SalIrestriction site. The amplified DNA fragment was digested with XbaI andSalI then ligated to XbaI and SalI cut pDSRα2. The resultant plasmid,pDSRα2 mpl ligand 1-174 was used for mammalian cell expression. Thesequence of the resulting gene (including the signal peptide) is shownin FIG. 2.

Plasmid DNA containing mpl ligand 1-174 was digested with XbaI and SalIrestriction enzymes, the resulting DNA fragments were subjected toagarose gel electrophoresis, and the 605 nt mpl ligand 1-174 DNAfragment was isolated from the gel using a GeneClean™ kit and proceduressupplied by the manufacturer (BIO 101, Inc.). Plasmid pDSRα2 asdescribed in WO 90/14363 (1990) was also digested with XbaI and SalIrestriction enzymes and the vector fragment was recovered. Ligation ofthe two fragments results in pDSRα2 (mpl ligand 1-174).

EXAMPLE 2 Expression of Mpl Ligand 1-174 in CHO Cells and Purification

Dihydrofolate reductase deficient (DHFR⁻) Chinese Hamster Ovary (CHO)cells were transfected with pDSRα2-mpl ligand 1-174. A 100 mm tissueculture dish was plated with 1×10⁶ CHO DHFR⁻ cells grown in CHO D-medium(DMEM, 10% Fetal bovine serum, 1% penicillin/ streptomycin/glutamine, 1%nonessential amino acids(Gibco) and 1% HT supplement (Gibco)) the daybefore transfection. Four transfections were performed. For eachtransfection, plasmid DNA (50 ug) was linearized by digesting with Pvu Iand Buffer H (Boehringer Mannheim). A DNA precipitate was then formedand added to the plates dropwise as per the Mammalian Cell TransfectionKit (Speciality Media). After 24 hours in a tissue culture incubator themedium was replaced with fresh CHO D- medium. Twenty four hours laterthe cells were split into 96 well tissue culture plates with 100 ul ofCHO select medium (D-MEM, 5% dialyzed fetal Bovine serum, 1%penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco))per well and transformants were selected. Medium was changed weeklyuntil colonies appeared. After two weeks, mpl ligand expression wasscreened for using the 32D cell proliferation assay described below (seeExample 9). Those clones expressing in excess of 1×10⁵ units/ml wereexpanded and frozen in cryogenic storage. One clone was expanded forroller bottle production and approximately 8 liters of conditionedmedium was produced.

Plasmid pDSRα2 containing mpl ligand 1-174 cDNA was transfected intoDHFR-deficient CHO cells as explained above. Two liters of serum-freeCHO cell conditioned medium (50% D-MEM, 50% HAMS-F12, 1%penicillin/streptomycin/glutamine, 1% nonessential amino acids(Gibco))from roller bottles seeded with CHO cells expressing mpl ligand 1-174was concentrated 15 fold using a 2L Amicon Model 2000 stirred cell and a10,000 dalton molecular weight cut-off membrane (YM10, Amicon).Forty-five milliliters of concentrated conditioned medium was thenloaded directly onto a 4 ml hu-MPL-X affinity column at a flow rate of0.4 ml/min using a Pharmacia FPLC. The affinity column was constructedby coupling 1.5-2.5 milligrams of Mpl-X (the soluble extra-cellulardomain of the mpl receptor) per milliliter of Pharmacia CNBr activatedSepharose resin as recommended by the manufacturer. After loading, thecolumn was washed with 16 ml of phosphate buffered saline (PBS; 10 mM Na. PO₄ pH 6.8/150 mM NaCl) and then 24 ml of 10 mM Tris, pH 8.0/1M NaCl.Mpl ligand (1-174) was eluted with 40 ml of 20 mM CAPS(3-[Cyclohexylamino]-1 propanesulfonic acid) pH 10.5/1M NaCl/5 mMCHAPS(3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate) in 6ml fractions. The second fraction yielded a single band on a 14% SDSgel. This material was concentrated and buffer exchanged against asaline solution of 0.9% NaCl and was biologically active in vitro and invivo. Other forms of CHO cell expressed mpl ligand were purified in asimilar manner.

EXAMPLE 3 In vivo Biolocrical Activity of Recombinant Human Mpl Ligand

Platelet counts from mice treated with various forms of mpl ligand weremeasured. CHO-derived mpl ligand 1-332, 1-174, 1-163, and 1-153 wereproduced and purified by Mpl-receptor affinity chromatography. E.coli-derived Met-Lys-mpl ligand 1-332, Met-Lys-mpl ligand 1-174,Met-Lys-mpl ligand 1-163 and Met-Lys-mpl ligand 1-153 were produced andpurified by conventional chromatography.

FIG. 4 shows platelet counts from mice treated with various forms of CHOcell-derived (solid lines) or E. coli-derived (dashed lines) recombinanthuman mpl ligand. Normal, female Balb/c mice were injectedsubcutaneously with the indicated concentration of mpl ligand for 5consecutive days. Test bleeds from a small lateral cut in a tail veinwere collected 24 hours after the last injection. Blood cell analyseswere performed with a Sysmex electronic blood cell analyzer (BaxterDiagnostics, Inc. Irvine, Calif.). Data are represented as the mean ofdeterminations of 4 animals, +/- standard error of the mean. Other bloodcell parameters such as total white blood cell counts or red blood cellcounts were not affected by these treatments (data not shown).

The results indicate that CHO cell expressed forms of mpl ligand have anincreased in vivo activity relative to the same forms of mpl ligandproduced in E. coli. As described in Example 6, the CHO cell expressedforms of mpl ligand all contain N and/or O-linked carbohydrate and theE. coli expressed mpl ligand forms do not. This indicates that thecarbohydrate enhances the in vivo activity of mpl ligand. The increasedin vivo activity conferred by the carbohydrate may be a result ofincreased circulatory half life, increased stability or a combination ofboth.

EXAMPLE 4 Construction of Mpl ligand Analogs N2-N15

Procedures for generating additional glycosylation sites for mpl ligandare described below.

The following oligonucleotide primers were synthesized for use in invitro mutagenesis to prepare analogs N2-N14 (see Table 1 for thestructures of these analogs):

    ______________________________________                                        N2 - CCCATGTCAATCACAGCAGACT                                                                             SEQ ID NO.:5                                        N3 - CTTCACAGCAACCTGAGCCAGT                                                                                                   SEQ ID NO.:6                  N4 - CAGTGCAACGAGACCCACCCTTTG                                                                                   SEQ ID NO.:7                                N5 - GCCTACAAATGTCACGCTGCCTGCT                                                                                 SEQ ID NO.:8                                 N6 - CCCACTTGTAACTCATCCCTC                                                                                         SEQ ID NO.:9                             N7 - CAACTGAACGCCACTTGTCTCTCA                                                                                   SEQ ID NO.:10                               N8 - ACTTGTCTCAACTCCACCCTGGGGGA                                                                               SEQ ID NO.:11                                 N9 - CTCCTGGGGAACCTTTCTGGA                                                                                         SEQ ID NO.:12                            N10 - GACCACAAATCACACCGATCCCAAT                                                                                 SEQ ID NO.:13                               N11 - ACCCTTTGTCTACAAATGTCACGCTGCCTGCT                                                                   SEQ ID NO.:14                                      N12 - TCTCTCAAACCTCACGGGGGAGCTT                                                                                 SEQ ID NO.:15                               N13 - TGGAAAAATCAGACGGAGGAGAC                                                                                     SEQ ID NO.:16                             N14 - TGGAGGAGAACAAGACACAGGACAT                                                                                 SEQ ID NO.:17                               ______________________________________                                    

To construct m13mpl8 mpl ligand 1-174, the gene of FIGS. 2A-2B wasintroduced into XbaI and SalI restriction enzyme digested m13mp18 DNA.Single stranded DNA was recovered from supernatants of E. coli strainRZ1032 infected by m13mp18(mpl ligand 1-174) as described by Kunkel etal., Methods in Enzymol. 154: 367 (1987) and Messing, Methods inEnzymol. 101: 20 (1983). For in vitro mutagenesis approximately 0.5 μgof single-stranded DNA and 0.125 pmole of one of the synthetic primersdescribed above were mixed with 6 μl of buffer (250 mM Tris pH 7.8, 50mM MgCl₂, 50 mM dithiothreitol and 1% Bovine serum albumin(BSA-Pharmacia)). The primers were previously kinased with ATP and T4polynucleotide kinase prior to addition. For annealing of the primer tothe template, the reaction volume was adjusted to 10 μl with water, themixture was heated to 65° C. for 5 minutes and then allowed to cool toroom temperature. For the elongation reaction 2.5 μl of each of dTTP,DATP, dGTP and dCTP and 1 ml ATP (all at 10 μM) were added, followed by1 μl (1 unit) of E. coli DNA polymerase (Klenow fragment) and 1 μl (1unit) of T4 DNA ligase. The mixture was then incubated overnight at 14°C. and used to transform E. coli JM 109 (Yanisch-Perron et al. Gene 33,103 (1985)) as described (Messing, supra).

To identify mutant clones by differential hybridization, plaques onnutrient agar were transferred to Gene Screen filters (New EnglandNuclear). The DNA was cross-linked to filters by irradiating them in aUV Stratalinker Model 1800 using the auto cross-link mode (Stratagene).They were then incubated for one hour in 6× SSC(0.9M NaCl/0.09MNa·citrate) containing 1% SDS at 60° C. For the hybridization, theoligonucleotide primer above (8 pmoles) was end-labeled with T4polynucleotide kinase and γ ³² P-labeled ATP and incubated with thefilters overnight in 6× SSC, 0.5% SDS and 125 ug/ml herring sperm DNA.The hybridization temperatures were chosen according to estimates ofoligonucleotide melting points. Generally the hybridization temperaturewas approximately 10° C. less than the melting point. The next day, thefilters were washed two times with 6× SSC/1% SDS at hybridizationtemperature followed by two washes with 6× SSC at hybridizationtemperature and subjected to autoradiography. If necessary, the filterswere then washed with 6× SSC at increasing temperatures until little orno hybridization was detected to plaques having the wild-type mpl ligandcDNA sequence. Clones that gave positive hybridization signals underthese conditions were identified and retransfected into JM109 to isolatea pure clone. Dideoxy chain termination sequence analysis indicated thatthe mutations were present.

Double stranded ml3 mpl ligand 1-174 DNAs carrying the desired changeswere recovered from JM109 transfected cells with QIAGEN kits (ChatsworthCalif.) using methods supplied by the manufacturer. The DNAs weredigested with XbaI and SalI and the 605 bp mpl ligand DNA fragments wereisolated. pDSRα2 was digested with XbaI and SalI. The vector fragmentwas isolated and ligated to the mpl ligand fragments above. Recombinantplasmids were identified by restriction analysis. The resulting plasmids(designated mpl ligand 1-174-NX where NX is the analog number) containDNA encoding mpl ligand analogs having altered amino acid residues atthe indicated positions. The resultant plasmids were then sequencedagain to confirm the presence of the desired mutations.

The analog N15 was constructed that had two additional N-linkedglycosylation sites at positions 30 and 120. PDSRα2 mpl ligand 174-N4containing Asn30 and Thr32 mutations was digested with XbaI and PstIrestriction enzymes and the approximately 385 nt DNA fragment wasisolated. PDSRα2 mpl ligand 174-N10 containing Asn120 and Thr122mutations was digested with PstI and SalI restriction enzymes and theapproximately 220 nt DNA fragment was isolated. pDSRα2 was digested withXbaI and SalI. The vector fragment was isolated and ligated to the mplligand fragments above. This resulted in PDSRα2 mpl ligand 174-N15 thatcontains Asn30, Thr32, Asn120 and Thr122 substitutions.

These general procedures were used to construct the mpl ligand analogsshown in Table 1. The DNA sequence changes for each of the analogs areshown; otherwise the oligonucleotide primers used for mutagenesis hadsequences complementary to those of human mpl ligand.

                                      TABLE 1                                     __________________________________________________________________________    Analog/                                                                              Amino Acid          Sequence                                           Species No.                                                                                  Substitution                                                                                            Changes                              __________________________________________________________________________    N1     (1-174); Pro.sup.175 → Gly.sup.332 deleted                                                 CCA → TGA (stop codon)                      N2                  Leu.sup.22 → Asn.sup.22                                                                 CCT → AAT                         N3                  Arg.sup.25 → Asn.sup.25                                                                 AGA → AAC                         N4     Pro.sup.30, Val.sup.32 → Asn.sup.30 , Thr.sup.32                                           CCA, GTT → AAC, ACC                         N5     Pro.sup.38, Leu.sup.40 → Asn.sup.38 , Thr.sup.40                                           CCT, CTG → AAT, ACG                         N6     Leu.sup.86 → Asn.sup.86                                                                    CTC → AAC                                   N7     Gly.sup.82, Pro.sup.83 → Asn.sup.82, Ala.sup.83                                            GGA, CCC → AAC, GCC                         N8     Ser.sup.87, Leu.sup.89 → Asn.sup.87, Thr.sup.89                                            TCA, CTC → AAC, ACC                         N9     Gln.sup.92 → Asn.sup.92                                                                    CAG → AAC                                   N10    Ala.sup.120, Lys.sup.122 → Asn.sup.120, Thr.sup.122                                        GCT, AAG → AAT, ACC                         N11    Pro.sup.36, Pro.sup.38, Leu.sup.40 →                                                       CCT, CCT, CTG →                                    Ser.sup.36, Asn.sup.38, Thr.sup.40                                                                TCT, AAT, ACG                                      N12    Ser.sup.88 Leu.sup.90 → Asn.sup.88, Thr.sup.90                                             TCC, CTG → AAC, ACG                         N13    Thr.sup.53, Met.sup.55 → Asn.sup.53, Thr.sup.55                                            ACC, ATG → AAT, ACG                         N14    Thr.sup.58, Ala.sup.60 → Asn.sup.58, Thr.sup.60                                            ACC, GCA → AAC, ACA                         N15    Pro.sup.30, Val.sup.32, Ala.sup.120, Lys.sup.122                                                  CCA, GTT, GCT, AAG →                               Asn.sup.30, Thr.sup.32, Asn.sup.120, Thr.sup.122                                                  AAC, ACC, AAT, ACC                                 __________________________________________________________________________     Note: Analogs N2-N15 are synonymously referred to herein as analogs 2-15.     Further, as used herein, for example, [Asn.sup.22 ] mpl ligand means that     an asparagine has been substituted for the amino acid at position 22 in       the particular mpl ligand species being considered, which is preferably a     human sequence having at least amino acids 7-151 of FIGS. 1A1C (inc1uding     the preferred human mpl ligand sequences set forth herein above). Thus,       substitution of an asparagine residue for a leucine residue at position 2     of mpl ligand 1-174 (human sequence) yields an mpl ligand analog that may     be represented by [Asn.sup.22 ] mpl ligand 1-174.                        

Note: Analogs N2-N15 are synonymously referred to herein as analogs2-15. Further, as used herein, for example, [Asn²² ] mpl ligand meansthat an asparagine has been substituted for the amino acid at position22 in the particular mpl ligand species being considered, which ispreferably a human sequence having at least amino acids 7-151 of FIGS.1A-1C (including the preferred human mpl ligand sequences set forthherein above). Thus, substitution of an asparagine residue for a leucineresidue at position 22 of mpl ligand 1-174 (human sequence) yields anmpl ligand analog that may be represented by [Asn²² ] mpl ligand 1-174.

Plasmids designated pDSRα2 1-174-NX (where NX is the analog number) wereconstructed by inserting mpl ligand DNA into pDSRα2. The expressionvector pDSRα2 is generally described in WO 90/14363(1990). pDSRα2 mplligand 1-174-NX plasmids were made by digestion of pDSRα2 with XbaI andSalI. The vector fragment was isolated and ligated to the approximately605 bp fragments containing the desired sequences.

EXAMPLE 5 Expression of Mpl Ligand and Mpl Ligands N1-N15 in COS Cells

cDNA clones of human mpl ligand and mpl ligand analogs described inTable 1 were transferred into COS-1 cells (ATCC No. CRL-1650) byelectroporation. COS-1 cells were harvested from semi-confluent dishes,washed with medium (Dulbecco's modified essential medium containing 10%fetal bovine serum and 1% L-glutamine/penicillin/streptomycin (IrvineScientific)) and resuspended at 6×10⁶ cells/ml. One half ml of cells wastransferred to a 0.2 cm electroporation cuvette (Bio-Rad) andelectroporated with a BTX Electroporation System Electrocell Manipulator600 at 650 uF and 130 volts on the low voltage setting with 50 μg ofplasmid DNA encoding the mpl ligand analog. The electroporated cellswere plated on 100 mm tissue culture dish in 10 ml of medium. Twelve totwenty four hours after plating the medium was replaced with 10 ml offresh medium. The conditioned medium was collected 3 to 5 days afterelectroporation.

EXAMPLE 6 Characterization of Mpl Ligand and Mpl Ligands N1-N15

A. Determination of Carbohydrate Addition

A volume of supernatant containing approximately 30-60 ng mpl ligand ormpl ligand analog from COS cells transfected with mpl ligand analogcDNAs as described in Example 5 was immunoprecipitated overnight at roomtemperature with a rabbit anti-mpl ligand polyclonal antibody. In somecases where expression was low, a maximum volume of approximately 8-9 mlwas used for immuno-precipitation. The antibody was raised to mpl ligand1-163 that had been expressed and purified from E. coli. Thirty μl of1:1 Protein A-Sepharose in phosphate buffered saline (PBS) containing0.1% sodium azide was added to the immunoprecipitate and allowed toincubate for one hour at room temperature. The samples were centrifuged,washed with PBS and resuspended in SDS sample Buffer (0.125 M Tris-HClpH 6.8/4% SDS/20% glycerol/10% β-mercaptoethanol/0.001% bromophenolblue). The samples were analyzed by 12% SDS-polyacrylamide gelelectrophoresis, transferred to nitrocellulose and subjected to Westernanalysis as described (Burnette et al., Anal. Biochem. 112: 195-203(1981); Elliott et al., Gene 79: 167-180 (1989)) using a mouse anti-mplligand monoclonal antibody raised to a synthetic mpl ligand peptide(e.g., corresponding to amino acid residues 47-62 of FIG. 1A). The mplligand containing bands were visualized using an ECL kit (Amersham).

FIG. 5 shows that COS cell supernatants from cells transfected withanalogs N4, N7 and N10 DNA revealed increased size compared to humansequence mpl ligand 174 (N1). FIG. 6 shows that COS cell supernatantsfrom cells transfected with N13, N14 and N4 DNA also had increased sizecompared to human sequence mpl ligand. This increased size is indicativeof an additional N-linked carbohydrate chain. N15 contains twoadditional N-linked glycosylation sites. FIG. 6 indicates that thisanalog has material with a size greater than analogs containing only 1additional N-linked glycosylation. The sizes of the proteins wereestimated from their mobility on SDS-PAGE relative to protein standardsof known molecular weight. The estimated sizes of the larger bandscalculated from FIG. 6 are shown in Table 2. This result indicates thatN15 contains 2 additional N-linked chains. Western blot analyses ofother selected analogs are also shown in FIG. 6.

                  TABLE 2                                                         ______________________________________                                        N-Linked Carbohydrate Estimates                                                                                No. of Potential                                                  Molecular   N-Linked                                     Mpl Ligand                                                                              Molecular  Weight Shift(Da)                                                                          Chains                                       Analog (1-174)                                                                          Weight (Da)                                                                              (Over Native)                                                                             (@ 4 KDa/Site)                               ______________________________________                                        N1 (Native)                                                                             23500        0         0                                             N4       28700      5200        1                                             N7       27200      3700        1                                            N10       27200      3700        1                                            N13       26700      3200        1                                            N14       28700      5200        1                                            N15       33500      10000       2                                            ______________________________________                                    

An experiment was performed to show that the increased size of mplligand analogs is due to N-linked carbohydrate. COS cell conditionedmedium containing mpl ligand was immunoprecipitated and washed with PBSas described above. To each tube was then added 10 μl 0.5% SDS and eachsample was boiled for 3 minutes. Then the following components wereadded: 10.8 μl of 0.5M NaPO₄ pH 8.6, 5 ml of 7.5% nonidet P40 and 3 ulof 250 unit/ml N-glycanase (Genzyme). N-glycanase treatment removesN-linked carbohydrate. Samples were incubated for 6 hours at 37° C. Thereaction was stopped by the addition of SDS-PAGE sample buffer and thensubjected to SDS-PAGE Western analysis (12% acrylamide) using ananti-mpl ligand monoclonal antibody and an anti-mouse ECL WesternDetection Kit (Amersham) as described above. An analysis of N-linkedchains using this method is shown in FIG. 7 for human mpl ligand and mplligand analogs. Following treatment with N-glycanase the mobility onWestern blot for N4, N7 and N10 was reduced to that of N1. As expected,treatment of N1 with N-glycanase had no effect on mobility because Nihas no N-linked glycosylation sites. These results indicate that theincreased size observed is due to addition of N-linked carbohydrate.

B. Analysis of O-Linked Carbohydrate on Mpl Ligand

To analyze the contribution of O-linked carbohydrate to human mplligand, various forms of the protein were purified from CHO cellconditioned media as described above. Each form received +/- treatmentwith O-glycanase (Glycopeptide alpha-N-acetylgalactos-aminidase, OxfordGlycoSystems). O-glycanase removes O-linked carbohydrate fromglycoproteins. The E. coli expressed version of each form was used as anunglycosylated control. To resolve the difference in molecular weightcontributed by O-linked carbohydrate, it was necessary to remove anyN-linked carbohydrate first. Since the full length version, mpl ligand1-332, contains N-linked carbohydrate, the CHO cell expressed fulllength samples received N-glycanase(peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) treatmentas described above for COS cell expressed mpl ligand analogs, exceptthat the N-glycanase treatment was an overnight incubation.

Before proceeding with the O-glycanase treatment on full length (1-332)mpl ligand, the pH range of the sample was adjusted to pH 6.0-pH 7.0with 1/15 volume of 100 mM acetic acid, pH 2.2. One microgram of proteinwas denatured by boiling for 3 minutes in SDS and incubated at 37° C.for 60 minutes with 1 U/ml neuraminidase (sialidase, from Arthrobacterurefaciens, Boehringer Mannheim) in 1 mM calcium acetate, pH 6.8 and 20mM sodium phosphate, pH 6.8.

Subsequent treatment with O-glycanase was done by adding 5 mU of enzymein a final volume of 100 ul, followed by an overnight incubation at 37°C. Proteins (0.2 ug/lane) were separated by SDS-PAGE (15% acrylamide).Following transfer to 0.2 um nitrocellulose and overnight incubationwith anti-mpl ligand polyclonal antibody the mpl ligand proteins werevisualized using an anti-rabbit ECL Western Detection Kit (Amersham).

FIG. 3 shows a Western blot of four different forms of human mpl ligand.Full length mpl ligand 1-332 is represented in lanes 1-3, mpl ligand1-174 lanes 4-6, mpl ligand 1-163 lanes 7-9, and mpl ligand 1-153 lanes10-12. Treatment with neuraminidase and O-glycanase, shown in lanes2,5,8, and 11, reduced the molecular weight to that of unglycosylatedmaterials, lanes 3,6,9, and 12. In every case the mobility increased tothat of the unglycosylated version expressed in E. coli. These resultsindicate that the larger sized bands, in lanes 1,4,7, and 10 are due toO-linked carbohydrate. The molecular weight of each of the bands wasestimated by comparing their mobilities to proteins of known molecularweight.

As seen in Table 3 which shows estimated molecular weights of thedifferent proteins, the apparent shift in mobility could account for asmany as 14 O-linked carbohydrate chains (assuming 950 daltons/chain) formpl ligand 1-332, 9 chains for mpl ligand 1-174, 4 chains for mpl ligand1-163, and 2 chains for mpl ligand 1-153. The sample run in lane 2 isfull length mpl ligand 1-332. It would appear that this protein wasdegraded, possibly due to extended incubation in glycoenzymes at 37° C.Therefore, the E. coli expressed unglycosylated version in lane 3 wasused to calculate the approximate molecular weight of O-linkedcarbohydrate added to CHO cell expressed mpl ligand 1-332.

These results are consistent with the presence of carbohydrate on allthe CHO expressed forms of mpl ligand tested. The presence of O-linkedcarbohydrate was confirmed for CHO cell expressed mpl ligand 1-332,1-174, and 1-163 by direct analysis of monosaccharide composition.Sialic acids, GalNAc and Gal were released from glycoproteins by acidhydrolysis. The monosaccharides were detected by high pressure anionexchange chromatography and pulsed amperometric detection. All threesugars were detected in each of the forms of mpl ligand. This result isindicative of the presence of sialic acid containing O-linkedcarbohydrate. This data correlates with the in vivo data as seen in FIG.4 where CHO cell expressed forms of mpl ligand were all more active invivo than the equivalent forms expressed in E. coli. Thus, the presenceof carbohydrate enhances the in vivo activity of mpl ligand.

                  TABLE 3                                                         ______________________________________                                        O-Linked Carbohydrate Calculations                                            Mpl   O-Glycanase          Molecular                                                                            # of Potential                              Ligand                                                                              Treatment  Molecular Weight O-Linked Chains                             Form  (+/-)      Weight (Da)                                                                             Shift  (@ 950 Da/Chain)                            ______________________________________                                        1-332 -          54200     13600  14                                          "     E. coli version                                                                          40600                                                        1-174 -          24600     8600   9                                           "     +          16000                                                        1-163 -          18400     3900   4                                           "     +          14500                                                        1-153 -          15200     2300   2                                           "     +          12900                                                        ______________________________________                                    

EXAMPLE 7 Mpl Ligand ELISA Assay

Polyclonal antibody production--New Zealand White rabbits werehyperimmunized over a period of three months with recombinant human mplligand 1-163 produced in E. coli. Antisera from six rabbits exhibitinghigh antibody titers were pooled and specific anti-mpl ligand antibodieswere affinity purified.

Affinity purification--Recombinant human mpl ligand 1-163 was covalentlyattached to Actigel-ALD (Sterogene Bioseparations, Inc.) according tothe manufacturer's instructions. An aliquot of the rabbit antisera poolwas added to the mpl ligand affinity gel, and the slurry was agitatedgently on a rocker platform overnight a 4-8° C. Unbound serum proteinswere washed from the gel bed with PBS and specifically bound anti-mplligand antibodies were then eluted with ImmunoPure Gentle Ag/Ab ElutionBuffer (Pierce Chemical Co.) Recovered antibodies were dialyzed againstseveral changes of PBS, then the antibody solution was concentrated inan Amicon stirred cell ultrafiltration unit and the resultant antibodyconcentrate was the source of specific anti-mpl ligand antibodiessubsequently used for well coating and enzyme conjugate preparations.

ELISA reagents--Immulon 4 Removawell Strips (Dynatech Laboratories,Inc.) were coated with affinity purified rabbit anti-mpl ligandantibodies. Affinity purified antibodies were diluted in 0.1 M sodiumbicarbonate (freshly prepared pH about 8.2) to a concentration of 2.5ug/ml. Each well received 100 ul of antibody and the plates wereincubated for 24 hrs at room temperature in a sealed and humidifiedchamber. Then, 200 ul of a blocking solution consisting of 1% fetalbovine serum 5% sucrose in TEN (50 mM Tris 7.4/10 mM EDTA/150 mM NaCl)was added to each well and plates were incubated and additional 24 hrsat room temperature in a sealed and humidified chamber. Combined coatingand blocking solutions were removed from the wells. An additionalovercoating/blocking step was included: 300 ul of SuperBlock BlockingBuffer in PBS (Pierce Chemical Co.) was added to each well. Afterstanding at room temperature for about 5 min. this solution was removedand the wells were allowed to air dry at room temperature for 24 hrs.The coated wells were stored in sealed plastic bags at 4-8° C. untilused in the mpl ligand ELISA.

Affinity purified anti-mpl ligand antibodies from a rabbit antisera poolwere covalently coupled to horseradish peroxidase (HRPO) for use as thesignal generating antibody. The affinity purified antibodies werederivatized with iminothiolane HCl (Fluka Chemical Corp.). Separately,HRPO was derivatized with N-succinimidyl 6-maleimidocaproate (FlukaChemical Corp.). The two activated proteins were combined to permitcovalent coupling. The reaction mixture was then chromatographed down aFPLC Superose 6 (Pharmacia) column to isolate the antibody:HRPOconjugate of the desired molecular weight (i.e. about 200 kD). Fractionscontaining the desired conjugate were combined and concentrated in aCentricon 30 (Amicon Division, W.R. Grace & Co.) and stored as a 50%glycerol solution at -20° C. This anti-mpl ligand Ab:HRPO concentratewas diluted into 2% fetal bovine serum in PBS for use in the ELISA. Thefinal concentration of conjugate used in the ELISA was 250-500 ng/ml.

Recombinant human mpl ligand 1-163 produced in E. coli cells, was usedfor the preparation of standards. This mpl ligand was diluted into 2%fetal bovine serum (Sigma Chemical Co.) in TEN buffer containing 0.05%thimerosal as a preservative. Standards prepared contained 1.0, 0.5,0.25, 0.125 and 0.062 ng/ml mpl ligand.

Assay-100 ul of mpl ligand standards or samples was added to wells thenincubated for 18-24 hrs at room temperature in a sealed and humidifiedchamber. The well contents and residual solution were then removed andthe wells washed once with wash solution (0.05% Tween 20 in TEN buffer).Anti-mpl ligand Ab:HRPO conjugate solution (100 ul) was added to eachwell and then incubated for 2 hrs at room temperature in a sealed andhumidified chamber. The contents of wells were removed then washed 4times with 0.05% Tween 20 in TEN buffer.

For color development, 100 ul of TME/peroxide substrate solution(Kirkegaard & Perry Solutions A & B mixed 1:1) was added and incubatedfor 20 min at room temperature. The reaction was stopped by addition of100 ul stop solution (0.5 N sulfuric acid) and the absorbance was readat 450 nm on microtiter plate reader. Concentrations of mpl ligand insamples were calculated from a standard curve generated by using a curvefit program.

EXAMPLE 8 Biological Activity of Mpl Ligand 1-174 Analogs in aShort-Term Liquid Culture Megakaryocyte Assay

Analogs of mpl ligand 1-174 were prepared as described above and assayedfor their ability to stimulate the growth of megakaryocytes in liquidculture. CD34 selected cells isolated from human leukapheresis units(Nichol et al., Stem Cells 12: 494-505 (1994)) were plated at 2×10⁵ /mlin culture medium (IMDM/1% Pen-Strep Glutamine/1% Non-essential AminoAcids/1% MEM Na-Pyruvate/1% MEM Vitamins/10% deionized BSA/10% normalhuman AB plasma/10 uM alpha-thiacylglycerol/20 ug/ml L-Asparagine). Inaddition, 1.5 ul of COS-1 conditioned medium containing mpl ligand(1-174) or mpl ligand 1-174 analog was added to each well. The finalvolume was 15 ul in Terasaki-style microtiter tissue culture plates(Vangard International). Cells were incubated at 37° C. for eight daysin humidified boxes in 5% CO₂, fixed directly to the culture wells with1% glutaraldehyde, and then incubated with a monoclonal antibodycocktail consisting of anti-GPIb, anti-GPIIb, (Biodesign) and anti-GPIb(Dako, Carpinteria, Calif.). The immune reaction was developed with astreptavidin-β-galactosidase detection system (HistoMark, Kirkegaard andPerry). Megakaryocytes, identified by the darker color (blue in actualphotographs), appear in FIGS. 8A-8D.

Panels A and D of FIGS. 8A-8D are the positive and negative controlsrespectively. The well pictured in panel A received 37.5 pg of wild typempl ligand 1-174 COS-1 conditioned medium and shows substantialmegakaryocyte growth. Panel D received 1.5 ul of COS-1 mock conditionedmedium and shows no growth. Panels B and C of FIGS. 8A-8D are mpl ligand1-174 analogs N7 and N10 respectively. Panel B received 9.0 pg of mplligand COS-1 conditioned medium while panel C received 27 pg and bothshow excellent megakaryocyte growth.

This experiment indicates that the analogs of mpl ligand tested arecapable of stimulating the growth of human megakaryocytes in vitro.

EXAMPLE 9 Biological Activity of Mpl Ligand 1-174 Analogs in an In VitroCell Proliferation Assay

Analogs of mpl ligand 1-174 were prepared as described above and assayedfor their ability to stimulate the proliferation of 32D-mpl cells. Toconstruct 32D-mpl cells, the full length human mpl receptor sequence(Vigon, I., et al., PNAS 89: 5640-5644 (1992)) was subcloned into anexpression vector containing the transcriptional promoter of MoloneyMurine Sarcoma virus. Six ug of this construct and 6 ug of anamphotrophic retroviral packaging construct (Landau, N. R., Littman, D.R., Journal of Virology 66: 5110-5113 (1992)) were transfected into3×10⁶ 293 cells using a CaPO₄ mammalian transfection kit (Stratagene).The same cells were retransfected after 2 days and again after 4 days.The day after the last transfection the 293 cells were cocultivated withthe IL-3 dependent murine cell line (32D, clone 23; Greenberger et al.,PNAS 80: 2931-2936 (1983)). After 24 hours, the 32D cells were rescuedand banded in a BSA gradient (Path-o-cyte; Miles Inc.). Cells wereexpanded in 1 ng/ml murine IL-3 and then were selected for growth in 20%APK9 serum (Bartley et al., Cell 77: 1117-1124 (1994). Cells were sortedfor cell surface expression of receptor by FACS using a polyclonalrabbit antipeptide (MPL) serum. These cytokine dependent murine 32D-mplcells are responsive to mpl ligand. 32D-MPL cells were grown in MEMmedium containing 10% Fetal Clone II Serum (Hyclone Laboratories) and1.0 ng/ml muIL3 to a cell density of 1×10⁶ cells/ml. Cells werecollected by centrifugation (approx. 500×G) and washed twice in growthmedium lacking muIL3 and resuspended at 1×10⁵ cells/ml.

An extended twelve point mpl ligand standard curve was prepared usingmpl ligand 1-163 and ranges from 5000 to 1 pg/ml. A volume of 100 ul ofeach dilution of standard mpl ligand or assay sample was added toappropriate wells of a 96 well microtiter tissue culture platecontaining 100 ul of resuspended cells (10,000 cells/well) and incubatedin a humidified incubator at 37° C. and 10% CO₂. After 48 hours, 40 ulof MTS reagent (Aqueous Non-Radioactive Cell Proliferation Kit, Promega)was added to each well and 14-18 hours later the plates were read on aplate reader at 490 nM. The in vitro activity in samples was calculatedfrom a dose response curve for each sample. One unit was defined as theamount of mpl ligand in each sample required to give 50% of maximalstimulation. Specific activity was calculated by dividing the biologicalactivity in units/ml by the mpl ligand concentration in ng/ml asdetermined by mpl ligand ELISA.

The specific biological activity of mpl ligand analogs transfected andexpressed in COS cells is shown in Table 4. The effect of the amino acidsubstitutions on carbohydrate addition is also summarized. Purifiedhuman sequence mpl ligand has an in vitro activity that was 200-300unit/ng as determined by the above-mentioned assays. It is apparent fromTable 4 that mpl ligand analogs containing additional N-linkedcarbohydrate are expressed as well as native sequence mpl ligand evenwhen they contain additional carbohydrate chains (as determined inExample 6, Section A) e.g., N4 and N10. Both of these analogs retainedfull in vitro biological activity also. Therefore the mpl ligand analogscontaining N-linked carbohydrate can be expressed normally in mammaliancells and they can have normal or enhanced in vitro biological activity.

                  TABLE 4                                                         ______________________________________                                        Mpl                                                                           Ligand                             In Vitro                                                                            Specific                             Form                Number of      Activity                                                                            Activity                             (Amino              N-linked Elisa (units/                                                                             (units/                              Acid                chains   (ng/ml)                                                                             ml)   ng)                                  Length)                                                                              Sequence     (a)      (b)   (c)   (d)                                  ______________________________________                                        MOCK   NONE         0        <0.08  <10  <125                                 N1 (174)                                                                             Native       NA       25    5375  215                                  N1 (174)                                                                             Native       0        31.4  8800  280                                  N1 (174)                                                                             Native       0        31.75 NA    NA                                   N2 (174)                                                                             N22          0        NA    NA    NA                                   N3 (174)                                                                             N25          NA       1.85   636  344                                  N4 (174)                                                                             N30T32       1        38    8830  232                                  N4 (174)                                                                             N30T32       1        24    NA    NA                                   N5 (174)                                                                             N38T40       0        1.2   <10    <8                                  N6 (174)                                                                             N86          0        0.44  <10   <22                                  N7 (174)                                                                             N82A83       0 to 1   6     2660  443                                  N7 (174)                                                                             N82A83       0 to 1   4.7   3080  655                                  N9 (174)                                                                             N92          0        10.5  1970  188                                  N10 (174)                                                                            N120T122     1        20.4  5943  291                                  N10 (174)                                                                            N120T122     1        33.7  9690  288                                  N11 (174)                                                                            S36N38T40    NA       <0.625                                                                               <10  <16                                  N11 (174)                                                                            S36N38T40    0        1.3    <10   <8                                  N13 (174)                                                                            N53T55       0 to 1   67    18000 269                                  N14 (174)                                                                            N58T60       0 to 1   17.9  4850  271                                  N15 (174)                                                                            N30T32N120T122                                                                             0 to 2   26    6420  247                                  ______________________________________                                         NOTES                                                                         (a) The number of additional Nlinked chains was estimated based upon the      mobility of the analog polypeptides in SDS gels as described in Example 6     (b) Quantities of mpl ligand analogs in CHO cell supernatants were            determined by ELISA assay as described in the Examples.                       (c) In vitro activity was determined by measuring stimulation of thymidin     uptake in 32D cells dependent on mpl ligand for growth.                       (d) Ratio of in vitro activity of mpl ligand analog as measured by            proliferation assays to amount of mpl ligand analog measured by mpl ligan     ELISA.                                                                        N.A. Not available.                                                      

EXAMPLE 10 Expression in CHO cells and Purification of Mpl Ligand 1-174,N4 and N15

pDSRα2 containing mpl ligand 1-174, N4 and N15 cDNA was transfected intoDHFR-deficient CHO cells using the protocol described in Example 2 withthe following modifications.

One transfection was performed for each analog. Three weeks after thetransfection, mpl ligand expression was screened by mpl ligand ELISA.Three expressing clones for each form were frozen in cryogenic storage.The highest expressing clone for each analog was expanded for rollerbottle production. For N4, 7.4 liters of conditioned medium (50% D-MEM,50% HAMS-F12, 1% penicillin/streptomycin/glutamine, 1% nonessentialamino acids (Gibco)) was produced and for N15, 4.6 liters of conditionedmedium was produced.

Serum-free CHO cell conditioned medium from roller bottles seeded withCHO cells expressing mpl ligand 1-174 (2.9 L), N4 (7.4 L), N15 (4.4 L)was concentrated 12-, 19-, and 12- fold respectively using a S1Y10(10,000 dalton molecular weight cut-off) Amicon Spiral ultrafiltrationcartridge. One hundred fifty milliliters of concentrated conditionedmedium was then loaded directly onto a 3.3 ml hu-MPL-X (receptor)affinity column at a flow rate of 0.3 ml/min. The affinity column wasconstructed by coupling 1.0-1.5 milligrams of Mpl-X (the solubleextra-cellular domain of the Mpl receptor) per milliliter of PharmaciaCNBr activated Sepharose resin as recommended by the manufacturer. Afterloading, the column was washed with 30 ml of phosphate buffered saline(PBS; 10 mM NaPO₄ pH 6.8/150 mM NaCl) and then 60 ml of 10 mM Tris, pH8.0/1M NaCl/1 mM CHAPS. Mpl ligand 1-174 was eluted with 30 ml of 20 mMCAPS (3-[Cyclohexylamino]-1 propanesulfonic acid) pH 10.5/1M NaCl/1 mMCHAPS(3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate).

Fractions were neutralized by adding 0.6 mL 1M Tris pH 7.0 to eacheluted fraction. SDS-PAGE analysis showed an apparent "bleeding" of1-174 mpl ligand during the 10 mM Tris, pH 8.0/1M NaCl 1 mM CHAPS wash.Elution fractions were analyzed by SDS-PAGE. Those fractions containingmpl ligand 1-174 were pooled. This affinity purification was thenmodified and repeated with the following changes: 0.5 mL/min load andelution, and the removal of the 10 mM Tris, pH 8.0/1M NaCl/1 mM CHAPSwash.

All fractions containing the single mpl ligand band were concentratedusing a YM10 (10,000 dalton molecular weight cut-off) membrane in a 50mL stirred cell, switching to a centricon device. This 0.5 mLconcentrate was loaded directly onto a PBS equilibrated PharmaciaSuperdex 200 HR 10/30 gel filtration column at 0.25 mL/min collecting0.25 mL fractions. All eluted fractions containing a single mpl ligandband (based on SDS-PAGE analysis) were pooled.

Other forms (N4 and N15) of CHO cell expressed mpl ligand were purifiedin a similar manner (two affinity purifications pooled and run on oneSuperdex 200 gel filtration column).

EXAMPLE 11 Determination of Carbohydrate Addition for CHO Cell ExpressedN4 and N15

In order to determine whether N-linked carbohydrate was contained in thempl ligand forms expressed in CHO cells, conditioned medium was analyzedby SDS-PAGE Western blot as described in Example 6 with the followingmodifications.

CHO D- Conditioned medium from roller bottles was used. Samples wereloaded into the Centricon-10 centrifugal concentrators (Amicon, Beverly,Mass.) and were spun at 6000 RPM for one hour in a Beckman J2-HScentrifuge using a fixed angle rotor (JA 20.1). A volume of concentratedsample containing approximately 100 ng of the mpl ligand analog wasloaded on a SDS PAGE gel together with SDS sample buffer (described inExample 6.) E. coli expressed mpl ligand MK 1-174 containing nocarbohydrate was also loaded. FIG. 9 shows differences in mobility thatcorrelate with the expected amount of carbohydrate. The fastest mobilityspecies, Met-Lys (1-174) E. coli mpl ligand, was followed by mpl ligand1-174 (CHO), N4 (CHO), and N15 (CHO) in succession. See FIG. 9. The mostlikely explanation for size increases relative to unglycosylated mplligand is additional O-linked carbohydrate on mpl ligand 1-174 (CHO),additional O-linked carbohydrate and one additional N-linkedoligosaccharide on N4 (CHO), and additional O-linked carbohydrate andtwo additional N-linked oligosaccharides on N15 (CHO).

In order to establish that the increase in molecular weight was indeeddue to the addition of N-linked carbohydrate chains, the samples weretreated with N-glycanase to remove any N-linked carbohydrate asdescribed in Example 6. Each sample contained approximately 100 ng ofmpl ligand analog purified from conditioned medium.

Following treatment with N-glycanase the mobility of N4 (CHO) and of N15(CHO) was reduced to that of mpl ligand 1-174 (CHO). Treatment of mplligand MK 1-174 (E. coli) or mpl ligand 1-174 (CHO) with N-glycanase didnot affect mobility since neither form was expected to contain anyN-linked carbohydrate. Comparison of N-glycanase treatment versus notreatment shows that the size difference for N4 corresponds to the sizeof one N-linked carbohydrate chain and the size difference for N15corresponds to the size of two carbohydrate chains. Thus addition ofN-linked glycosylation sites for these two mpl ligand forms resulted inadditional N-linked carbohydrate when these species were expressed inCHO cells. See FIG. 10.

EXAMPLE 12 In vitro Biological Activity of Mpl Ligand Analogs made inCHO cells

Purified mpl ligand and analogs expressed and purified from in CHO cellsor E. coli cells were analyzed for in vitro biological activity usingthe factor dependent cell line 32D-MPL and assay described in Example 9except activity was calculated from a curve using mpl ligand 1-332produced in CHO cells as standard. The specific in vitro biologicalactivities of the various forms are shown in Table 5. It is apparentfrom this Table that the mpl ligand analogs containing additionalcarbohydrate, which are expressed in CHO cells have in vitro biologicalactivity.

                  TABLE 5                                                         ______________________________________                                        IN VITRO ACTIVITY OF MPL LIGANDS                                                                        IN VITRO                                            MPL LIGAND     No. N-Linked                                                                             ACTIVITY                                            FORM           Chains     U/mg X10E6                                          ______________________________________                                        MK174 (E. coli)                                                                              0          13                                                  1-163 (CHO)    0          86                                                  1-174 (CHO)    0          85                                                  N4 (CHO)       1          60                                                  N15 (CHO)      2          92                                                  1-332 (CHO)    6          41                                                  ______________________________________                                    

EXAMPLE 13 In vivo Biological Activity of Mpl Ligand Analogs

Platelet counts from mice treated with various forms of mpl ligand weremeasured and the results are presented in FIG. 11. CHO-derived mplligand 1-332, 1-174, N4, and N15 were produced and purified bympl-receptor affinity chromatography. E. coli-derived Met-Lys-mpl ligand1-174, was produced and purified by conventional chromatography. Theindicated concentration of each form was administered subcutaneouslyinto normal, female Balb/c mice once daily for 5 days. Test bleeds froma small lateral cut in a tail vein were collected 24 hours after thelast injection. Blood cell analyses were performed with a Sysmexelectronic blood cell analyzer (Baxter Diagnostics, Inc. Irvine,Calif.). Data are represented as the mean of determinations of 4animals, +/- standard error of the mean. Other blood cell parameterssuch as total white blood cell counts or red blood cell counts were notaffected by these treatments (data not shown).

All the forms stimulated increases in platelet counts. However theactivities of the different forms varied. The relative in vivo activitywas mpl ligand MK 1-174 (E. coli) <mpl ligand 1-174 (CHO) <N4 (CHO) <mplligand 1-332 (CHO) <N15 (CHO). The results indicate that addition ofnon-naturally occurring N-linked carbohydrate results in increased invivo activity. It indicates further that increases in the amount ofcarbohydrate result in proportional increases in in vivo activity.

EXAMPLE 14 Construction of Mpl Ligand Analogs and Truncations N16-N40 byOverlap PCR

Analogs N16 to N40 (see Table 6 for the structures of these analogs)were constructed by overlap PCR (polymerase chain reaction) using aprotocol adapted from Cheng et al., PNAS 91, 5695 (1994). Typically oneto two mutations were introduced in each construction.

The following oligonucleotide primers were synthesized for use toprepare analogs N16-N40:

    __________________________________________________________________________    5'40  F CCCTCTAGACCACCATGGAACTGACTGAATTGCTCCTC                                                           SEQ ID NO.: 18                                     3'40  R (1-174) CCCGTCGACTCAGAGCTCGTTCAGTGTG                                                                  SEQ ID NO.: 19                                N16 - 3'40  R CCCGTCGACTCACTCCAACAATCCAGAAG                                                              SEQ ID NO.: 20                                     N17 - 3'40  R CCCGTCGACTTATCTGGCTGAGGCAGTGA                                                              SEQ ID NO.: 21                                     N18 - F CACGTCCTTAACAGCAGCCTGAGCCAGTG                                                                            SEQ ID NO.: 22                             N18 - R CACTGGCTCAGGCTGCTGTTAAGGACGTG                                                                            SEQ ID NO.: 23                             N19 - F CCCTTTGCCTAACGGTTCCCTGCTGCCTGCTGT                                                                    SEQ ID NO.: 24                                 N19 - R ACAGCAGGCAGCAGGGAACCGTTAGGCAAAGGG                                                                    SEQ ID NO.: 25                                 N20 - F TGCCTACACCTAACCTGTCGCCTGCTGTGGA                                                                        SEQ ID NO.: 26                               N20 - R TCCACAGCAGGCGACAGGTTAGGTGTAGGCA                                                                        SEQ ID NO.: 27                               N21 - F GGAAAACCAATATGTCGGAGACCAAGGCACA                                                                        SEQ ID NO.: 28                               N21 - R TGTGCCTTGGTCTCCGACATATTGGTTTTCC                                                                        SEQ ID NO.: 29                               N22 - F TGGGAGAATGGAACACCACGATGGAGGAGACC                                                                      SEQ ID NO.: 30                                N22 - R GGTCTCCTCCATCGTGGTGTTCCATTCTCCCA                                                                      SEQ ID NO.: 31                                N23 - F AAAACCCAGATGAACGAGACGACCAAGGCACA                                                                      SEQ ID NO.: 32                                N23 - R  TGTGCCTTGGTCGTCTCGTTCATCTGGGTTTT                                                                    SEQ ID NO.: 33                                 N24 - F CCCAGATGGAGAACACCTCGGCACAGGACAT                                                                        SEQ ID NO.: 34                               N24 - R ATGTCCTGTGCCGAGGTGTTCTCCATCTGGG                                                                        SEQ ID NO.: 35                               N25 - F CACGGGGACAAAACGGAACCACTTGCCTCTCA                                                                      SEQ ID NO.: 36                                N25 - R TGAGAGGCAAGTGGTTCCGTTTTGTCCCCGTG                                                                      SEQ ID NO.: 37                                N26 - F CAGGGCAGGAACACATCTCACAAGGATCCCA.                                                                      SEQ ID NO.: 38                                N26 - R TGGGATCCTTGTGAGATGTGTTCCTGCCCTG                                                                        SEQ ID NO.: 39                               N27 - F GGGCAGGACCAACGCTAGCAAGGATCCCAAT                                                                         SEQ ID NO.: 40                              N27 - R ATTGGGATCCTTGCTAGCGTTGGTCCTGCCC                                                                        SEQ ID NO.: 41                               N29 - F pair1 CAGTGCAACGAGTCCCACCCTTGG                                                                           SEQ ID NO.: 42                             N29 - R pair1 CAAAGGGTGGGACTCGTTGCACTG                                                                          SEQ ID NO.: 43                              N29 - F pair2 GACCACAAATCACTCCGATCCCAA                                                                           SEQ ID NO.: 44                             N29 - R pair2 TTGGGATCGGAGTGATTTGTGGTC                                                                           SEQ ID NO.: 45                             N30 - F GTCCCCACCAACACCTCTCTAGTCCTC                                                                                 SEQ ID NO.: 46                          N30 - R GAGGACTAGAGAGGTGTTGGTGGGGAC                                                                                 SEQ ID NO.: 47                          N31 - 3'40  R CCCGTCGACTCACTTCAGAAGCCCAGAGCCAGT                                                            SEQ ID NO.: 48                                   N36 (1) - F GAAAACCCAGAACGAGACCACCAAGGCACAG                                                                  SEQ ID NO.: 49                                 N36 (1) - R CTGTGCCTTGGTGGTCTCGTTCTGGGTTTTC                                                                  SEQ ID NO.: 50                                 N36 (2) - F CACCAAGGCACAGGACATTCTGGGAG                                                                         SEQ ID NO.: 51                               N36 (2) - R CTCCCAGAATGTCCTGTGCCTTGGTG                                                                         SEQ ID NO.: 52                               N37 - F GAAAACCCAGATGAACGAGACCAAGGCACAG                                                                       SEQ ID NO.: 53                                N37 - R CTGTGCCTTGGTCTCGTTCATCTGGGTTTTC                                                                       SEQ ID NO.: 54                                N38 - F GTCCCCACCAACACCACTCTAGTCCTC                                                                               SEQ ID NO.: 55                            N38 - R GAGGACTAGAGTGGTGTTGGTGGGGAC                                                                             SEQ ID NO.: 56                              __________________________________________________________________________     F = Forward                                                                   R = Reverse                                                              

Constructions that introduce one new glycosylation site were performedin two successive steps. In step 1, two reactions were performed using 4different oligonucleotides. These oligos included a 5' forward primer, areverse mutagenic primer, a forward mutagenic primer(usuallycomplementary to the reverse mutagenic primer) and a reverse 3' primer.The reverse 3' primer contained sequences that introduced stop codonsfollowed by SalI restriction sites. Stop codons were introduced atpositions 175, 184, 192, and 200. Thus, forms of lengths 1-174, 1-183(N16), 1-191 (N17), and 1-199 (N31) could be made. PCR1 used templateDNA (pDSRα2 containing mpl ligand 1-174 sequences or full length mplligand 1-332 sequences), the 5' forward primer and the reverse mutagenicprimer. PCR2 used template DNA, the 3' reverse primer and the forwardmutagenic primer. The two PCR reactions were then performed and theamplified DNA fragments were separated by agarose gel electrophoresis.Small pieces of agarose containing DNA fragments of the correct sizewere excised from the gel.

The DNA fragments from PCR1 and PCR2 were combined together and a thirdPCR reaction was performed using only the 5' forward and 3' reverseprimers. Thus, a full length DNA segment containing the desiredmutations inserted into mpl ligand was amplified.

The amplified fragments were again separated by agarose gelelectrophoresis, the correct sized DNA fragment was purified using aGeneclean™ kit and procedures supplied by the manufacturer (Bio 101,Inc.). The purified DNA was digested with XbaI and SalI, then it waspurified again using the Geneclean™ kit. The fragment was then ligatedinto XbaI and SalI cut pDSRα2. Ligated DNA was precipitated with 2volumes of ethanol in 0.3M NaOAc pH 5.2 in the presence of carrier tRNAand transformed into E. coli. Clones were tested by restriction analysisand agarose gel electrophoresis to identify those containing thecorrectly sized DNA inserts. Purified plasmid DNA was then prepared andthe mpl ligand insert was sequenced to confirm the presence of thedesired mutations and to ensure that no additional amino acid changeswere introduced.

In several cases, two or more mutations were combined simultaneously,i.e., see N29, N33, N34, N35, N39 and N40. This could be done byintroducing a new substitution into DNA already containing a change. Forexample, N33 was made by introducing the N23 changes into N15. In thiscase the procedure above was performed by using N23 mutagenic primersand the N15 template DNA.

In another strategy, two changes could be introduced simultaneously intotemplate DNA. The template DNA could contain natural sequences or couldcontain sequences encoding mpl ligand forms already containing changes.In these cases step 1 involved 3 PCR reactions and 6 oligos. The oligosincluded a 5' forward primer, 2 pairs of forward and reverse mutagenicprimers, and a reverse 3' primer. Each pair of primers was complementaryto each other and contained sequences designed to introduce one newglycosylation site.

PCR1 included template DNA, the 5' forward primer and the reversemutagenic primer from pair 1. PCR2 included template DNA, the forwardmutagenic primer from pair 1 and the reverse mutagenic primer from pair2 where pair 2 primers are 3' to pair 1 primers. PCR3 included templateDNA, the forward mutagenic primer from pair 2 and the reverse 3' primer.

DNA fragments from each PCR reaction were separated by agarose gelelectrophoresis and excised as before. The 3 DNA fragments were thencombined together and amplified by PCR again using only the 5' forwardand 3' reverse primers.

The DNA segment encoding the entire gene of interest with sequencescontaining two new glycosylation sites was then purified, cut with XbaIand SalI, and ligated into XbaI and SalI cut pDSRα2 as before.

Multiple mutations could also be combined by performing the PCRreactions on templates already containing mutations. For example, N39was made by introducing N36 and N38 changes into N15 template DNA. Thiswas done using a different set of primers (N36(2)) than that used tomake N36 (N36(1)). See primers set forth above. Both sets of primersintroduced the same mutation.

Longer mpl ligand forms could also be made. Thus, N40 was made in asimilar manner to N39 except the 3' reverse primer in PCR 3, (step 1)and the PCR primer in step 2 was the primer used to make N31. Thisprimer introduces a stop codon at position 200 followed by a SalIrestriction site. In addition, the template DNA used for PCR 3 containedsequences encoding full length mpl ligand (1-332).

The typical PCR reaction mix contained: 4 ul each of forward and reverseprimers (5 pm/ul), 1 ul template (50 ng), 10 ul of 5X LP buffer (100 mMTricine pH 8.7/25% glycerol/425 mM KOAc), 10 ul dNTP stock (1 mM each ofdATP, dTTP, dCTP, dGTP), 0.8 ul rtTh polymerase (Perkin Elmer; 2.5U/ul), and 2 ul Vent polymerase (NEB; 0.01 U/ul after 1:100 freshdilution in 1X LP buffer). H₂ O was added to bring the final volume to50 ul. All the components were added together in the order shown and thePCR was started when the temperature during the first cycle was abovethe 60° C. by adding 1 ul of 50 mM MgOAc. Reaction conditions were: 2cycles of 94° C., 10 sec/45° C., 1 min/68° C., 5 min followed by 25cycles of 94° C., 10 sec/55° C., 1 min/68° C., 5 min.

These general procedures were used to construct the mpl ligand analogsand truncations N16 to N40 shown in Table 6. The DNA sequence changesfor each of the forms are shown.

                                      TABLE 6                                     __________________________________________________________________________    MPL LIGAND ANALOGS HAVING SITES                                               FOR N-LINKED CARBOHYDRATE CHAINS                                              Analog/                                                                              Amino Acid                                                             Species No.                                                                                  Substitution  Sequence Changes                                 __________________________________________________________________________    N16    (1-183); Thr.sup.184 → Gly.sup.332 deleted                                                   ACA → TGA (stop codon)                    N17         (1-191); Thr.sup.192 → Gly.sup.332 deleted                                              ACT → TAA (stop codon)                    N18           His.sup.23, Arg.sup.25 → Asn.sup.23,                                                       CAC, AGA → AAC, AGC                  N19             Thr.sup.37, Pro.sup.38, Val.sup.39 →                                                            ACA, CCT, GTC                                                  Asn.sup.37, Gly.sup.38, Ser.sup.39                                                         → AAC, GGT, TCC                 N20          Val.sup.39, Leu.sup.41 → Asn.sup.39,                                                        GTC, CTG → AAC, TCG                  N21           Gln.sup.54, Glu.sup.56 → Asn.sup.54,                                                       CAG, GAG → AAT, TCG                  N22          Lys.sup.52, Gln.sup.54 → Asn.sup.52,                                                        AAA, CAG → AAC, ACG                  N23            Glu.sup.57 → Asn.sup.55'(i), Thr.sup.57                                                   GAG → AAC (i), ACG                   N24           Glu.sup.57, Lys.sup.59 → Asn.sup.57,                                                      GAG, AAG → AAC, TCG                   N25           Leu.sup.81, Pro.sup.83 → Asn.sup.81,                                                       CTG, CCC → AAC, ACC                  N26          Thr.sup.118, Ala.sup.120 → Asn.sup.118,                                                  ACC, GCT → AAC, TCT                     N27          Thr.sup.119, His.sup.121 → Asn.sup.119,                                                  ACA, CAC → AAC, AGC                     N29          Pro.sup.30, Val.sup.32, Ala.sup.120, Lys.sup.122                                                   CCA, GTT, GCT, AAG →                                        Asn.sup.30, Ser.sup.32, Asn.sup.120,                                                    AAC, TCC, AAT, TCC                           N30         Ser.sup.163, Arg.sup.164 → Thr.sup.163,                                                  AGC, AGA → ACC, AAC                      N31       (1-199); Trp.sup.200 → Gly.sup.332 deleted                                                    TGG → TGA (stop codon)                N33        Pro.sup.30, Val.sup.32, Glu.sup.57, Ala.sup.120,                                                CCA, GTT, GAG, GCT, AAG                                 →               →                                                          Asn.sup.30, Thr.sup.32, Asn.sup.55'40 (i),                                                   AAC, TCC, AAC (i), ACG,                                                Asn.sup.120, Thr.sup.122                                                                         AAT, TCC                         N34        Pro.sup.30, Val.sup.32, Glu.sup.57, Ser.sup.163,                                                CCA, GTT, GAG, AGC, AGA                                 →              →                                                Asn.sup.30, Thr.sup.32, Asn.sup.55'40 (i), Thr.sup.57,                                               AAC, TCC, AAC (i), ACG,                                                    Thr.sup.163, Asn.sup.164                                                                         ACC, AAC                        N35          N4 + N23 + N30 + N31 (1-199)                                                                                 -- --                             N36          Met.sup.55, Glu.sup.57 → Asn.sup.55,                                                         ATG, GAG → AAC, ACC                 N37               Glu.sup.56 → Asn.sup.56                                                                              GAG → AAC              N38           Ser.sup.163, Arg.sup.164, Ser.sup.166                                                                    AGC, AGA, TCT                                             → Thr.sup.163, Asn.sup.164,                                                               → ACC, AAC, ACT                N39          N4 + N10 + N36 + N38 (1-174)                                                                                 -- --                             N40        N4 + N10 + N36 + N38 + N31 (1-199)                                                                           -- --                               __________________________________________________________________________

The symbol "(i)" in the above Table means that the referenced amino acidhas been inserted. For example, Glu⁵⁷ →Asn^(55')(i), Thr⁵⁷ (analog N23in Table 6) means that the Glu at position 57 has been replaced with aThr and, additionally, an Asn has been inserted just after the Met atposition 55, and the Asn has been numbered 55' so that subsequent aminoacids retain their previously assigned numbers.

Examples that include all changes from previous Examples are indicatedby the specific analog numbers joined by "+" signs. See analogs N35,N39, and N40. The lengths of the amino acid chains of these analogs areindicated parenthetically. Thus, analog N35 contains a combination ofall changes made for analogs N4, N23, N30 and N31. The changes indicatedfor N31 mean that analog N35 is 199 amino acids long. All analogs inTable 6 are 174 amino acids long, except where indicated to be adifferent length (or, in the cases where an amino acid has beeninserted, the total length will be increased by the number of insertedamino acids).

EXAMPLE 15 Characterization of Mpl Ligand Analogs and Truncations N16 toN40

A. Determination of Expression Level and in Vitro Biological Activity ofMpl Ligand Analogs

Species N16 to N40 were transfected into COS cells using either theelectroporation method (Example 5) or the CaPO4 method (Mammalian cellTransfection Kit; Specialty media). Cell free conditioned medium wascollected after 3-4 days, aliquoted and stored at -70° C. Expressionlevel was determined by ELISA assay as described in Example 7. Thesupernatants were also assayed for biological activity as described inExample 9 with one modification. The activities were calculated from astandard curve using purified CHO cell expressed mpl ligand 1-332 asstandard.

The results are shown in Table 7. As shown in Table 7 most of the mplligand analogs were expressed and secreted. Some of the analogs appearedto have increased secretion. Bioassays on these samples indicated thatthe specific activities for most were also comparable to unmodifiedforms. Some of the analogs contained multiple N-linked carbohydratechains (see below). This indicates that carbohydrate addition can resultin increased secretion and normal in vitro activity of the analogs.

                  TABLE 7                                                         ______________________________________                                        Mpl                                                                           Ligand                             In Vitro                                                                            Specific                             Form                Number of      Activity                                                                            Activity                             (Amino              N-linked Elisa (units/                                                                             (units/                              Acid                chains   (ng/ml)                                                                             ml)   ng)                                  Length)                                                                              Sequence     (a)      (b)   (c)   (d)                                  ______________________________________                                        N1 (174)                                                                             Native       0        28    3991  143                                  N15 (174)                                                                            N30T32N120T122                                                                             0-2      45    7003  156                                  N16 (183)                                                                            1-183        0        85    9276  NA                                   N17 (191)                                                                            1-191        NA       <0.3   11   NA                                   N18 (174)                                                                            N23S25       0        2       5   2.5                                  N19 (174)                                                                            N37G38S39    NA       <0.3  NA    NA                                   N20 (174)                                                                            N39S41       NA       <0.3   <10  NA                                   N21 (174)                                                                            N54S56       0-1      30    4380  146                                  N22 (174)                                                                            N52T54       0-1      2      856  428                                  N23 (174)                                                                            N55'(i)T57   1        11    1059   96                                  N24 (174)                                                                            N57S59       0        5.3    458   86                                  N25 (174)                                                                            N81T83       NA       0.22   123  559                                  N26 (174)                                                                            N118S120     NA       0.9    96   106                                  N27 (174)                                                                            N119S121     0        4.5    338   75                                  N29 (174)                                                                            N30S32N120S122                                                                             0-2      15    1627  108                                  N30 (174)                                                                            T163N164     0-1      128   15592 122                                  N31 (199)                                                                            1-199        at least 1                                                                             156   19000 122                                  N33 (174)                                                                            4 + 10 + 23  3        78    10057 129                                  N34 (174)                                                                            4 + 23 + 30  at least 2                                                                             112   13536 120                                  N35 (199)                                                                            34 + 31      4 or more                                                                              172   13112  76                                  N36 (174)                                                                            N55T57       0-1      48    5808  121                                  N37 (174)                                                                            N56          1        32    4504  141                                  N38 (174)                                                                            T163N164T166 0-1      25    3904  156                                  N39 (174)                                                                            N4 + N10 + N36 +                                                                           3 to 4   127   17661 139                                         N38                                                                    N40 (199)                                                                            N4 + N10 + N36 +                                                                           at least 5                                                                             134   19735 147                                         N38 + N31                                                              ______________________________________                                         NOTES                                                                         (a) The number of additional Nlinked chains was estimated according to th     mobility of the analog polypeptides in SDS gels.                              (b) Quantities of mpl ligand analogs in COS cell supernatants were            determined by EIA.                                                            (c) In vitro activity was determined by measuring proliferation of 32DMPL     cells which are dependent on mpl ligand for growth.                           (d) Ratio of in vitro activity of mpl ligand analog as measured by            proliferation assays to amount of mpl ligand analog measured by mpl ligan     ELISA.                                                                        i  Insertion                                                                  NA Not available                                                         

B. Determination of Carbohydrate Addition

The analogs shown in Table 6 were tested to see if they added N-linkedcarbohydrate using the procedures described in Example 6.

Some analogs (N21, N22, N30, N33, and N36) were also tested with amodified procedure. This was necessary because the monoclonal antibodyused to develop the Western blot was raised to a peptide including aminoacid residues 47 to 62, and some of the analogs described in Table 6contain substitutions that affected immunoreactivity with this antibody,e.g. N21. Therefore, to analyze these analogs the supernatants wereimmunoprecipitated using a monoclonal antibody raised in mice to E. colicell expressed mpl ligand 1-163.

Typically 15 ugs of antibody was used to immunoprecipitate 50 ng of mplligand analog. Western blots with immunoprecipitated material wereperformed as described in Example 6 except the immunoprecipitated bandswere visualized by incubating the blots with the rabbit anti-mpl ligandpolyclonal antibody (typically 1 ug/ml; raised to E. coli cell expressedmpl ligand 1-163) and an anti rabbit ECL kit (Amersham). The results ofthe various experiments are shown in Table 7. Some of the analogs hadincreased size indicative of the presence of N-linked carbohydrate (N21,N22, N23, N29, N30, N31, N33, N34, N35, N36, N38, N39, and N40). Asubset of these analogs had more than 1 N-linked chain, e.g., N29, N33,N34, N35, N39 and N40. These analogs were secreted at normal or higherlevels and had in vitro biological activity comparable to mpl ligand1-174. This indicates that multiple functional N-linked glycosylationsites can be introduced into mpl ligand without a deleterious effect oneither expression or biological activity.

To demonstrate that multiple oligosaccharide chains can be added to mplligand, various analogs expressed in COS cells were analyzed by Westernblot as described in Example 6. FIG. 12 shows that the mobility of theanalogs decreases with increasing numbers of added N-linkedglycosylation sites. Analogs with 4 new sites are shown, N39 and N40.The analogs with the most N-linked sites had the slowest mobility. Thisresult is observed with both 1-174 and 1-199 forms of mpl ligand. Thisindicates that at least 4 analogs can be combined together resulting innew analogs with multiple N-linked carbohydrate chains.

EXAMPLE 16 Comparison of Glycosylation Sites Containing Asn-X-Ser vs.Asn-X-Thr

N-linked glycosylation sites include either Asn-X-Thr or Asn-X-Ser whereX can be any one of the 20 naturally occurring amino acids except Pro.We wished to determine whether Ser or Thr is preferred in the thirdposition. Therefore, two sets of analogs with each set containing a mplligand glycosylation analog containing either a Ser or Thr at the thirdposition in the sequon were examined to see if there was an effect onpercent occupancy of the N-linked glycosylation sites. N15 contains 2Asn-X-Thr sites while N29 contains 2 Asn-X-Ser sites at exactly the samepositions. In a similar manner N30 contains an Asn-X-Ser while N38contains an Asn-X-Thr at the same position.

To compare these two sets of analogs, they were expressed in COS cellsand the secreted mpl ligand was subjected to Western analysis asdescribed in Example 6. FIG. 13 shows the results. N15 had asignificantly increased proportion of glycosylated mpl ligand ascompared to N29. In contrast, there was very little difference in theproportion of glycosylated and unglycosylated mpl ligand when N30 andN38 were compared. These results indicate that both Asn-X-Ser andAsn-X-Thr can be introduced into mpl ligand and that both can act assites for N-linked carbohydrate addition. In addition, in some cases theAsn-X-Thr sequon may be preferred (i.e., it may be more efficientlyglycosylated).

EXAMPLE 17 Construction of Mpl Ligand Analogs and Truncations N41-N43 byOverlap PCR

Analogs N41, N42 and N43 (see Table 8 for the structures of theseanalogs) were constructed by overlap PCR (polymerase chain reaction)using a protocol adapted from Cheng et al PNAS 91, 5695 (1994).

The following oligonucleotide primers were synthesized for use toprepare analogs N41-N43:

    __________________________________________________________________________    5'40  F CCCTCTAGACCACCATGGAACTGACTGAATTGCTCCTC                                                            SEQ ID NO.: 18                                    3'40  R (1-174) CCCGTCGACTCAGAGCTCGTTCAGTGTG                                                                  SEQ ID NO.: 19                                3'40  R (1-199) CCCGTCGACTCACTTCAGAAGCCCAGAGCCAGT                                                         SEQ ID NO.: 57                                    N41 - F TCCCCAGCAACACCTCTCTAGTC                                                                                         SEQ ID NO.: 58                      N41 - R AGGTGTTGCTGGGGACAGCTGTG                                                                                         SEQ ID NO.: 59                      N42 - F TCCCCAACAGAACCTCTCTAGTC                                                                                         SEQ ID NO.: 60                      N42 - R AGGTTCTGTTGGGGACAGCTGTG                                                                                         SEQ ID NO.: 61                      N37 - F GAAAACCCAGATGAACGAGACCAAGGCACAG                                                                         SEQ ID NO.: 53                              N37 - R CTGTGCCTTGGTCTCGTTCATCTGGGTTTTC                                                                         SEQ ID NO.: 54                              __________________________________________________________________________     F = Forward                                                                   R = Reverse                                                              

Mpl ligands N41 and N42 were constructed using the corresponding PCRprimer pairs above and SEQ ID NO. 18 and SEQ ID NO. 19 outside primersusing the 2-step PCR protocol described in Example 14. Step 2 for eachof the constructions used the 5' primer (SEQ ID NO. 18) and a 3' primerthat resulted in a 1-174 form (SEQ ID NO. 19).

N43 was constructed by simultaneously introducing by PCR 2 N-linkedsites into templates already containing other N-linked sites using the 2step protocol as described in Example 14. Three different DNA templateswere used in step 1 (N15, N10 and 1-332) in 3 separate PCR reactions.PCR reaction 1 used template N15 and SEQ ID NO. 18 and SEQ ID NO. 54primers. This reaction introduced the N37 substitution into a DNAsegment already containing the N4 substitution. PCR reaction 2 used N10template and SEQ ID NO. 53 and SEQ ID NO. 59 primers. This reactionintroduced N37 and N41 mutations into a DNA fragment already containingthe N10 substitution. PCR reaction 3 used 1-332 template and SEQ ID NO.58 and SEQ ID NO. 57 primers. This reaction introduced N41 substitutionsinto a DNA fragment containing the 1-199 C terminus. Each of the DNAfragments were purified by agarose gel electrophoresis. The 3 DNAfragments were then combined together in 1 tube and joined by performinga PCR using SEQ ID NO. 18 and SEQ ID NO. 57 primers. This resulted in aDNA fragment that contained N4, N37, N10, and N41 substitutions in anmpl ligand 1-199 form. The DNA fragment was then cloned into PDSRα2 asdescribed in Example 14.

The overlap PCR reaction conditions for N41, N42 and the first threereactions in step 1 for N43 were: 1 cycle 94° C., 4 min; 25 cycles of94° C., 10 sec/68° C., 2 min; 1 cycle 68° C., 5 min.

The final PCR reaction conditions for N43 were: 1 cycle 94° C., 4 min; 5cycles of 94° C., 10 sec/44° C., 30 sec; 25 cycles of 94° C., 10 sec/68°C., 2 min; 1 cycle 68° C., 5 min.

Mpl ligand analogs clones N41-N43 were initially screened by colony PCRto identify clones containing the correctly sized and type of DNAinsert. Transformed bacterial (E. coli) colonies were picked with asterile innoculation loop and resuspended in 1X Taq buffer (BoehringerMannheim). Taq polymerase was added at 1.25 U/50 ul reaction with afinal 0.2mM dNTP mix and final 0.2 um of two vector primers. The colonyPCR reaction conditions were 1 cycle 95° C.; 35 cycles of 94° C., 30sec/52° C., 30 sec/72° C., 30 sec; 1 cycle 72° C., 5 min. PCR productswere anayzed by gel electrophoresis and sequencing.

                  TABLE 8                                                         ______________________________________                                        MPL LIGAND ANALOGS HAVING SITES                                               FOR N-LINKED CARBOHYDRATE CHAINS                                              Analog/                                                                       Species                                                                                       Amino Acid                                                    No.           Substitution  Sequence Changes                                  ______________________________________                                        N41    Arg.sup.164 → Asn.sup.164                                                                  AGA → AAC                                   N42              Ser.sup.163 → Asn.sup.163                                                                    AGC → AAC                       N43        N4 + N10 + N37 + N41 + N31                                                                             -- --                                                                   (1-199)                                         ______________________________________                                    

EXAMPLE 18 Characterization of Mpl Ligand Analogs and Truncations N41 toN43

A. Determination of Expression Level and in Vitro Biological Activity ofMpl Ligand Analogs

Species N41 to N43 were transfected into COS cells using theelectroporation method described in EXAMPLE 5 with the followingchanges. 25 ug of plasmid DNA encoding the mpl ligand analog was used.The electroporated cells were plated on 100 mm tissue culture dish in 7ml of medium. The conditioned medium was collected 3 days afterelectroporation, aliquoted and stored at -70° C.

Expression level was determined by ELISA assay as described in Example7. The supernatants were also assayed for biological activity asdescribed in Example 9 with one modification. The activities werecalculated from a standard curve using purified CHO cell expressed mplligand 1-332 as standard.

The results are shown in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    Mpl Ligand                         Specific                                   Form                Number of In Vitro                                                                           Activity                                   (Amino              N-linked                                                                           Elisa                                                                              Activity                                                                           (units/                                    Acid                chains                                                                             (ng/ml)                                                                            (units/ml)                                                                         ng)                                        Length)                                                                             Sequence      (a)  (b)  (c)  (d)                                        __________________________________________________________________________     N1 (174)                                                                           Native        0     28   5400                                                                              193                                        N31 (199)                                                                           Native        at least 1                                                                         156  15000                                                                               96                                        N38 (174)                                                                           T163N164T166  0-1   55   6080                                                                              110                                        N40 (199)                                                                           N4 + N10 + N36 + N38 + N31                                                                  at least 5                                                                         134  18100                                                                              135                                        N41 (174)                                                                           N163          0-1  109  17500                                                                              161                                        N42 (174)                                                                           N164          0-1   51   9300                                                                              182                                        N43 (199)                                                                           N4 + N10 + N37 + N41 + N31                                                                  at least 5                                                                         153  30500                                                                              199                                        __________________________________________________________________________     NOTES                                                                         (a) The number of additional Nlinked chains was estimated according to th     mobility of the analog polypeptides in SDS gels.                              (b) Quantities of mpl ligand analogs in COS cell supernatants were            determined by EIA.                                                            (c) In vitro activity was determined by measuring proliferation of 32DMPL     cells which are dependent on mpl ligand for growth.                           (d) Ratio of in vitro activity of mpl ligand analog as measured by            proliferation assays to amount of mpl ligand analog measured by mpl ligan     ELISA.                                                                   

B. Determination of Carbohydrate Addition

The analogs shown in Table 8 were tested to see if they added N-linkedcarbohydrate. Approximately 0.5 ng mpl ligand or mpl ligand analog fromCOS cells transfected with the analog cDNAs were mixed with SDS samplebuffer containing β-mercaptoethanol and boiled. The samples wereanalyzed by 12% SDS-polyacrylamide gel electrophoresis, transferred tonitrocellulose and subjected to Western anlysis. Immunoprecipitatedbands were visualized by incubating the blots with the rabbit anti-mplligand polyclonal antibody (typically 1 ug/ml; raised to E. coli cellexpressed mpl ligand 1-163) and an anti rabbit ECL kit (Amersham).

FIG. 14 shows that 3 different variants of mpl ligand can beglycosylated even though they differ in location and sequence around theglycosylation site. N38 contains T163, N164, and T166 mutations. N41 hasonly the N164 mutation. This analog has an N-linked site because of anexisting Ser at position 166 resulting in the sequence Asn-X-Ser.Analogs N38 and N41 are glycosylated similarly. This indicates that somepositions in mpl ligand are tolerant of substantial change in sequenceyet can be glycosylated efficiently. As shown in Table 9, both analogsretain similar in vitro biological activity. In contrast, N42 has an Asn163 substitution. This analog contains an N-linked glycosylation sitebecause of an existing Thr at position 165 resulting in an Asn-X-Thrglycosylation site. It also is glycosylated but less efficiently thanN38 or N41.

FIG. 15 shows comparisons of mpl ligands N40 and N43. Both mpl ligandscontain 6 glycosylation sites. They differ in the number of amino acidchanges used to introduce N-linked sites (6 amino acid changes for N43vs. 9 for N40) as well as the locations of the sites. The mobility ongels of N43 is the same as that of N40. This indicates that both mplligands are glycosylated to similar extents. Both analogs also havesimilar in vitro bioactivity as indicated in Table 9. This indicatesthat different analogs can be combined together to generate new mplligands that have similar properties such as number of N-linked chainsas well as similar in vitro bioactivity.

While the invention has been described in what is considered to be itspreferred embodiments, it is not to be limited to the disclosedembodiments, but on the contrary, is intended to cover variousmodifications and equivalents included within the spirit and scope ofthe appended claims, which scope is to be accorded the broadestinterpretation so as to encompass all such modifications andequivalents.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 61                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1342 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 36..1094                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 99..1094                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 36..98                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #ACT GAA TTG        53A GACACCCCGG CCAGA ATG GAG CTG                          #   Met Glu Leu Thr Glu Leu                                                   20                                                                            - CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA AG - #G CTA ACG CTG TCC AGC          101                                                                          Leu Leu Val Val Met Leu Leu Leu Thr Ala Ar - #g Leu Thr Leu Ser Ser           # 1                                                                           - CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CT - #C AGT AAA CTG CTT CGT          149                                                                          Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Le - #u Ser Lys Leu Leu Arg           #              15                                                             - GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CA - #G TGC CCA GAG GTT CAC          197                                                                          Asp Ser His Val Leu His Ser Arg Leu Ser Gl - #n Cys Pro Glu Val His           #         30                                                                  - CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT GT - #G GAC TTT AGC TTG GGA          245                                                                          Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Va - #l Asp Phe Ser Leu Gly           #     45                                                                      - GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GC - #A CAG GAC ATT CTG GGA          293                                                                          Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Al - #a Gln Asp Ile Leu Gly           # 65                                                                          - GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GC - #A GCA CGG GGA CAA CTG          341                                                                          Ala Val Thr Leu Leu Leu Glu Gly Val Met Al - #a Ala Arg Gly Gln Leu           #                 80                                                          - GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CA - #G CTT TCT GGA CAG GTC          389                                                                          Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gl - #n Leu Ser Gly Gln Val           #             95                                                              - CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC CT - #T GGA ACC CAG CTT CCT          437                                                                          Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Le - #u Gly Thr Gln Leu Pro           #       110                                                                   - CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CC - #C AAT GCC ATC TTC CTG          485                                                                          Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pr - #o Asn Ala Ile Phe Leu           #   125                                                                       - AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CG - #T TTC CTG ATG CTT GTA          533                                                                          Ser Phe Gln His Leu Leu Arg Gly Lys Val Ar - #g Phe Leu Met Leu Val           130                 1 - #35                 1 - #40                 1 -       #45                                                                           - GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CC - #A CCC ACC ACA GCT GTC          581                                                                          Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pr - #o Pro Thr Thr Ala Val           #               160                                                           - CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AA - #C GAG CTC CCA AAC AGG          629                                                                          Pro Ser Arg Thr Ser Leu Val Leu Thr Leu As - #n Glu Leu Pro Asn Arg           #           175                                                               - ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GC - #C TCA GCC AGA ACT ACT          677                                                                          Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Al - #a Ser Ala Arg Thr Thr           #       190                                                                   - GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TT - #C AGA GCC AAG ATT CCT          725                                                                          Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Ph - #e Arg Ala Lys Ile Pro           #   205                                                                       - GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GA - #C CAA ATC CCC GGA TAC          773                                                                          Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu As - #p Gln Ile Pro Gly Tyr           210                 2 - #15                 2 - #20                 2 -       #25                                                                           - CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA AC - #T CGT GGA CTC TTT CCT          821                                                                          Leu Asn Arg Ile His Glu Leu Leu Asn Gly Th - #r Arg Gly Leu Phe Pro           #               240                                                           - GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GA - #C ATT TCC TCA GGA ACA          869                                                                          Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro As - #p Ile Ser Ser Gly Thr           #           255                                                               - TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CA - #G CCT GGA TAT TCT CCT          917                                                                          Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gl - #n Pro Gly Tyr Ser Pro           #       270                                                                   - TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT AC - #G CTC TTC CCT CTT CCA          965                                                                          Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Th - #r Leu Phe Pro Leu Pro           #   285                                                                       - CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC CA - #C CCC CTG CTT CCT GAC         1013                                                                          Pro Thr Leu Pro Thr Pro Val Val Gln Leu Hi - #s Pro Leu Leu Pro Asp           290                 2 - #95                 3 - #00                 3 -       #05                                                                           - CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC CC - #T CTT CTA AAC ACA TCC         1061                                                                          Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pr - #o Leu Leu Asn Thr Ser           #               320                                                           - TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GG - #G TAAGGTTCTC AGACACTGCC       1114                                                                          Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gl - #y                               #           330                                                               - GACATCAGCA TTGTCTCGTG TACAGCTCCC TTCCCTGCAG GGCGCCCCTG GG - #AGACAACT       1174                                                                          - GGACAAGATT TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA TA - #CACAGGAC       1234                                                                          - TGAAAAGGGA ATCATTTTTC ACTGTACATT ATAAACCTTC AGAAGCTATT TT - #TTTAAGCT       1294                                                                          #              1342AGAG CAGCTAGCTC TTTGGTCTAT TTTCTGCA                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 353 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Glu Leu Thr Glu Leu Leu Leu Val Val Me - #t Leu Leu Leu Thr Ala         10                                                                            - Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Al - #a Cys Asp Leu Arg Val         #                 10                                                          - Leu Ser Lys Leu Leu Arg Asp Ser His Val Le - #u His Ser Arg Leu Ser         #             25                                                              - Gln Cys Pro Glu Val His Pro Leu Pro Thr Pr - #o Val Leu Leu Pro Ala         #         40                                                                  - Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gl - #n Met Glu Glu Thr Lys         #     55                                                                      - Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Le - #u Leu Glu Gly Val Met         # 75                                                                          - Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Le - #u Ser Ser Leu Leu Gly         #                 90                                                          - Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gl - #y Ala Leu Gln Ser Leu         #            105                                                              - Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Th - #r Thr Ala His Lys Asp         #       120                                                                   - Pro Asn Ala Ile Phe Leu Ser Phe Gln His Le - #u Leu Arg Gly Lys Val         #   135                                                                       - Arg Phe Leu Met Leu Val Gly Gly Ser Thr Le - #u Cys Val Arg Arg Ala         140                 1 - #45                 1 - #50                 1 -       #55                                                                           - Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Se - #r Leu Val Leu Thr Leu         #               170                                                           - Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Le - #u Glu Thr Asn Phe Thr         #           185                                                               - Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Le - #u Lys Trp Gln Gln Gly         #       200                                                                   - Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gl - #n Thr Ser Arg Ser Leu         #   215                                                                       - Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile Hi - #s Glu Leu Leu Asn Gly         220                 2 - #25                 2 - #30                 2 -       #35                                                                           - Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Ar - #g Thr Leu Gly Ala Pro         #               250                                                           - Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Se - #r Leu Pro Pro Asn Leu         #           265                                                               - Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pr - #o Pro Thr Gly Gln Tyr         #       280                                                                   - Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Th - #r Pro Val Val Gln Leu         #   295                                                                       - His Pro Leu Leu Pro Asp Pro Ser Ala Pro Th - #r Pro Thr Pro Thr Ser         300                 3 - #05                 3 - #10                 3 -       #15                                                                           - Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gl - #n Asn Leu Ser Gln Glu         #               330                                                           - Gly                                                                         - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 605 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 12..596                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 75..596                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 12..74                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #GTG GTC ATG CTT CTC      50CT GAA TTG CTC CTC                                #Thr Glu Leu Leu Leu Val Val Met Leu Leu                                      10                                                                            - CTA ACT GCA AGG CTA ACG CTG TCC AGC CCG GC - #T CCT CCT GCT TGT GAC           98                                                                          Leu Thr Ala Arg Leu Thr Leu Ser Ser Pro Al - #a Pro Pro Ala Cys Asp           #           5  1                                                              - CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TC - #C CAC GTC CTT CAC AGC          146                                                                          Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Se - #r His Val Leu His Ser           #     20                                                                      - AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TT - #G CCT ACA CCT GTC CTG          194                                                                          Arg Leu Ser Gln Cys Pro Glu Val His Pro Le - #u Pro Thr Pro Val Leu           # 40                                                                          - CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TG - #G AAA ACC CAG ATG GAG          242                                                                          Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Tr - #p Lys Thr Gln Met Glu           #                 55                                                          - GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GT - #G ACC CTT CTG CTG GAG          290                                                                          Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Va - #l Thr Leu Leu Leu Glu           #             70                                                              - GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA CC - #C ACT TGC CTC TCA TCC          338                                                                          Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pr - #o Thr Cys Leu Ser Ser           #         85                                                                  - CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CT - #C CTC CTT GGG GCC CTG          386                                                                          Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Le - #u Leu Leu Gly Ala Leu           #    100                                                                      - CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CA - #G GGC AGG ACC ACA GCT          434                                                                          Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gl - #n Gly Arg Thr Thr Ala           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TT - #C CAA CAC CTG CTC CGA          482                                                                          His Lys Asp Pro Asn Ala Ile Phe Leu Ser Ph - #e Gln His Leu Leu Arg           #               135                                                           - GGA AAG GTG CGT TTC CTG ATG CTT GTA GGA GG - #G TCC ACC CTC TGC GTC          530                                                                          Gly Lys Val Arg Phe Leu Met Leu Val Gly Gl - #y Ser Thr Leu Cys Val           #           150                                                               - AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC AG - #C AGA ACC TCT CTA GTC          578                                                                          Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Se - #r Arg Thr Ser Leu Val           #       165                                                                   #            605   AG CTC TAGGTCGAC                                           Leu Thr Leu Asn Glu Leu                                                           170                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 195 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Glu Leu Thr Glu Leu Leu Leu Val Val Me - #t Leu Leu Leu Thr Ala         10                                                                            - Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Al - #a Cys Asp Leu Arg Val         #                 10                                                          - Leu Ser Lys Leu Leu Arg Asp Ser His Val Le - #u His Ser Arg Leu Ser         #             25                                                              - Gln Cys Pro Glu Val His Pro Leu Pro Thr Pr - #o Val Leu Leu Pro Ala         #         40                                                                  - Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gl - #n Met Glu Glu Thr Lys         #     55                                                                      - Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Le - #u Leu Glu Gly Val Met         # 75                                                                          - Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Le - #u Ser Ser Leu Leu Gly         #                 90                                                          - Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gl - #y Ala Leu Gln Ser Leu         #            105                                                              - Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Th - #r Thr Ala His Lys Asp         #       120                                                                   - Pro Asn Ala Ile Phe Leu Ser Phe Gln His Le - #u Leu Arg Gly Lys Val         #   135                                                                       - Arg Phe Leu Met Leu Val Gly Gly Ser Thr Le - #u Cys Val Arg Arg Ala         140                 1 - #45                 1 - #50                 1 -       #55                                                                           - Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Se - #r Leu Val Leu Thr Leu         #               170                                                           - Asn Glu Leu                                                                 - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                 22AGA CT                                                    - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #                 22CCA GT                                                    - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #                24ACCC TTTG                                                  - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #               25 CTGC CTGCT                                                 - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #21                CCCT C                                                     - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #                24GTCT CTCA                                                  - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #              26  CCCT GGGGGA                                                - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #21                CTGG A                                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #               25 GATC CCAAT                                                 - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #          32      TGTC ACGCTGCCTG CT                                         - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #               25 GGGG AGCTT                                                 - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #                23AGGA GAC                                                   - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #               25 ACAG GACAT                                                 - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #     38           GGAA CTGACTGAAT TGCTCCTC                                   - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..28)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #             28   ACTC AGCTGCCC                                              - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..29)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #            29    CACT CAGCTGCCC                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..29)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #            29    TATT CAGCTGCCC                                             - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #            29    GCCT GAGCCAGTG                                             - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..29)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #            29    CGGA CTCGGTCAC                                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #         33       TCCC TGCTGCCTGC TGT                                        - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..33)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #         33       AGGG ACGACGGACG ACA                                        - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #          31      GTCG CCTGCTGTGG A                                          - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #          31      CAGC GGACGACACC T                                          - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #          31      GGAG ACCAAGGCAC A                                          - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #          31      CCTC TGGTTCCGTG T                                          - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #          32      CACG ATGGAGGAGA CC                                         - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..32)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #          32      GTGC TACCTCCTCT GG                                         - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #          32      AGAC GACCAAGGCA CA                                         - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..32)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #          32      TCTG CTGGTTCCGT GT                                         - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #          31      CTCG GCACAGGACA T                                          - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #          31      GAGC CGTGTCCTGT A                                          - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #          32      AACC ACTTGCCTCT CA                                         - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..32)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #          32      TTGG TGAACGGAGA GT                                         - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #          31      CTCA CAAGGATCCC A                                          - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #          31      GAGT GTTCCTAGGG T                                          - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #          31      AGCA AGGATCCCAA T                                          - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                #          31      TCGT TCCTAGGGTT A                                          - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #                24ACCC TTGG                                                  - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..24)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #                24TGGG AAAC                                                  - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #                24GATC CCAA                                                  - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..24)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #                24CTAG GGTT                                                  - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #             27   CTCT AGTCCTC                                               - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..27)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #             27   GAGA TCAGGAG                                               - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..33)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #         33       ACTT CACTCAGCTG CCC                                        - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #          31      ACCA CCAAGGCACA G                                          - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                #          31      TGGT GGTTCCGTGT C                                          - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #              26  ATTC TGGGAG                                                - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..26)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #              26  TAAG ACCCTC                                                - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #          31      GAGA CCAAGGCACA G                                          - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..31)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #          31      CTCT GGTTCCGTGT C                                          - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #             27   CTCT AGTCCTC                                               - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..27)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                #             27   GAGA TCAGGAG                                               - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..33)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                #         33       ACTT CACTCAGCTG CCC                                        - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #                23TCTA GTC                                                   - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..23)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                #                23TTGT GGA                                                   - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                #                23TCTA GTC                                                   - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "nucleic acid"CRIPTION: /desc                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY:                                                       #(1..23)  (B) LOCATION: complement                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #                23TCTT GGA                                                   __________________________________________________________________________

What is claimed is:
 1. An analog of mpl ligand, wherein(a) said mplligand comprises a sequence of amino acids selected from the groupconsisting of amino acid sequences 7-151 through 1-332, inclusive of SEQID NO:2, (b) said analog of mpl ligand has at least one added N-linkedglycosylation site in said sequence of amino acids, (c) said analog ofmpl ligand has a biological activity of specifically stimulating orincreasing megakaryocytes or platelets, and (d) said at least one addedN-linked glycosylation site is selected from the group consistingof:(Asn164); (Asn163); and (Asn30, Thr32, Asn56, Asn120, Thr122,Asn164).
 2. An analog of claim 1, wherein the mpl ligand has an aminoacid sequence selected from the group consisting of:

    ______________________________________                                        mpl ligand 1-332                                                                           amino acids 1-332 of SEQ ID NO: 2                                mpl ligand 1-199                                                                           amino acids 1-199 of SEQ ID NO: 2                                mpl ligand 1-191                                                                           amino acids 1-191 of SEQ ID NO: 2                                mpl ligand 1-183                                                                           amino acids 1-183 of SEQ ID NO: 2                                mpl ligand 1-174                                                                           amino acids 1-174 of SEQ ID NO: 2                                mpl ligand 7-332                                                                           amino acids 7-332 of SEQ ID NO: 2                                mpl ligand 7-191                                                                           amino acids 7-191 of SEQ ID NO: 2                                mpl ligand 7-199                                                                           amino acids 7-199 of SEQ ID NO: 2                                mpl ligand 7-183                                                                           amino acids 7-183 of SEQ ID NO: 2 and                            mpl ligand 7-174                                                                           amino acids 7-174 of SEQ ID NO:
 2.                               ______________________________________                                    


3. An analog of claim 2, wherein the mpl ligand has an amino acidsequence consisting of amino acids 1-199 of SEQ ID NO:
 2. 4. An analogof mpl ligand according to claim 2 which is;(Asn30, Thr32, Asn56,Asn120, Thr122, Asn164) mpl ligand 1-199 of SEQ ID NO:2.
 5. An analogaccording to any one of claims 1-4, which is the product of expressionof an exogenous DNA sequence in a eukaryotic cell.
 6. An analogaccording to claim 5, wherein said eukaryotic cell is a mammalian cell.7. An analog according to claim 6, wherein said mammalian cell is CHO.8. A composition comprising an mpl ligand analog according to any one ofclaims 1-4 together with a pharmaceutically acceptable diluent, adjuvantor carrier.